Study of the cellular and molecular mechanism associated ... · paciente comigo! Quero ainda agradecer-te por toda a tua disponibilidade para me ajudares, para me ouvires a treinar

“Discovery consists of looking at the same thing as everyone else and thinking

something different.”

― Albert Szent-Györgyi

http://www.goodreads.com/author/show/2499827.Albert_Szent_Gy_rgyi

FCUP/ICBAS Study of the cellular and molecular mechanism associated with neonatal susceptibility to bacterial infections

I

Aknowledgments

Em primeiro lugar, como não podia deixar de ser, gostaria de agradecer ao meu

orientador, Pedro Madureira. Fui sem dúvida alguma uma privilegiada por te ter como

orientador e por poder fazer parte da tua investigação. Sei que tenho muito, mas mesmo

muito a agradecer-te, tanto que por mais que escrevesse ia-me sempre esquecer de

alguma coisa. Mas como tenho de ser sucinta, gostava de te agradecer principalmente

por teres confiado em mim e no meu trabalho, pela tua disponibilidade (mesmo para as

dúvidas de última hora!), por toda a ajuda na interpretação dos resultados, por todas as

“discussões” científicas (onde aprendi mesmo muito!) e por seres uma pessoa tão

metódica e rigorosa. Gostaria ainda de te agradecer por tudo aquilo que me ensinaste,

e por esse teu lado positivo tão característico, que nos enche de confiança! Obrigada

por me teres feito ver a ciência, a investigação com outros olhos, e me teres feito crescer

enquanto investigadora! Foi um privilégio trabalhar contigo e só tenho mesmo de te

agradecer pelo excelente investigador e orientador que és!

Gostaria ainda de agradecer à minha co-orientadora, a professora Paula

Ferreira. Em primeiro lugar agradeço o facto de me ter recebido no seu laboratório, no

seu grupo de investigação. Foi em grande parte pela forma entusiaste com que fala da

imunologia, com que fala do seu trabalho, da investigação, que me fez escolher este

tema! Sem dúvida, a professora é e sempre será uma referência não só como docente,

mas como investigadora.

Em terceiro lugar quero agradecer a toda a equipa da Immunethep, em especial

a três pessoas que foram cruciais durante este ano e cuja ajuda foi incansável: Ana

Marques, Marta Vieira e Liliana Curado. Ana, não há palavras para descrever a

importância que tiveste durante este ano! És sem dúvida uma das melhores pessoas

que conheci! Gostaria de te agradecer por tudo aquilo que me ensinaste (até

conseguiste colocar uma Bioquímica a fazer técnicas histológicas!), por toda a tua

paciência, pela disponibilidade, por todas as conversas e acima de tudo por acreditares

sempre nas minhas capacidades. Queria ainda agradecer-te pelas horas que

dispensaste ao me acompanhares nos primeiros passos na experimentação animal!

Fizeste-me perder o medo e acreditar que também sou capaz! Foste sem dúvida uma

pessoa fulcral ao longo deste ano, por isso a ti um grande, grande Obrigada!

À Marta gostaria, primeiro que tudo, de agradecer pela excelente pessoa que é.

Foste sem dúvida uma das pessoas mais importantes para o sucesso deste trabalho e

por isso tenho muito, mas mesmo muito a agradecer-te! Obrigada pelas inúmeras

explicações, pelo facto de estares sempre disponível para me ajudar e por teres aceite

II FCUP/ICBAS Study of the cellular and molecular mechanism associated with neonatal susceptibility to bacterial infections

o desafio de partilhar comigo a câmara de fluxo do biotério e da P2 em dias de

experiência (tu ententes!). Apesar de muito cansativo posso dizer-te que foi dos

melhores dias de experiência! És sem dúvida uma referência enquanto pessoa e

investigadora! À Liliana gostaria de agradecer toda a colaboração durante este ano, e

de igual forma, o facto de estar sempre disponível para me ajudar. És uma das pessoas

mais rigorosas e metódicas que já conheci, e com quem aprendi muito, muito mesmo!

À Cristiana, ao Tiago e à Carla agradeço toda a ajuda, colaboração durante este

ano. Podem acreditar que todos foram muito importantes para que este trabalho fosse

possível e que com cada um de vocês aprendi mais um bocado. Afinal, a ciência é feita

de partilha e de entreajuda!

Gostaria ainda de agradecer a todo o grupo “Immunobiology” do i3S e à

Encarnação por toda a ajuda durante este ano. Um agradecimento especial à Mariana

por ser uma pessoa incansável, sempre disponível para ajudar. Foi um privilégio

partilhar contigo um cantinho da câmara de culturas estéreis! Obrigada pelas inúmeras

vezes que me emprestaste a tua micropipeta multicanal. Sem ti as ELISAS teriam sido

uma tarefa difícil de cumprir!

Agradeço ainda ao grupo “Molecular Parasitology” pela disponibilidade sempre

demonstrada, pelos esclarecimentos de última hora e por toda a preocupação.

Um obrigada a todos os colegas (Ângela, Liliana, Inês…) que me acompanharam

durante estes dois anos de Mestrado. Obrigada pelos momentos de gargalhada, por

todas as conversas (científicas e não científicas). Obrigada por tornarem estes dois anos

(fora os três anteriores em que muitos de vocês já me aturaram!) tão inesquecíveis,

repletos de momentos únicos que vou sempre guardar com saudade, mas ao mesmo

tempo com o contentamento de os ter partilhado com vocês! Não posso adivinhar o que

o futuro reserva para cada um de nós, mas sei que independentemente de tudo, as

amizades verdadeiras vão permanecer e tudo continuará a ser como antes!

Um agradecimento especial à Inês Lopes pelo facto de ser uma grande amiga e

estar sempre disponível para me ajudar. Felizmente quis o destino que nos

cruzássemos no mesmo laboratório. Foi sem dúvida uma excelente oportunidade para

nos ficarmos a conhecer um pouco melhor, o que fez com que a nossa amizade

crescesse. Quero agradecer-te por tudo aquilo que me ensinaste, pelas longas

conversas, por todas as “discussões” científicas, por seres uma excelente parceira de

laboratório e por estares sempre disponível para me ouvir e para me ajudar! As nossas

aventuras no Mário Arala Chaves são memoráveis! Sabes que podes sempre contar

comigo, para tudo mesmo!

Por último, mas claro o mais importante, gostaria de agradecer às três pessoas

mais importantes da minha vida, a minha mãe, o meu pai e a minha irmã, Rita. Aos


III

meus pais gostaria de agradecer o facto de terem confiado sempre em mim e me terem

feito seguir atrás dos meus sonhos, mesmo quando me lembrava de seguir o curso mais

estranho do Mundo! Quando a Bioquímica começou a ser realmente um sonho que

queria concretizar apoiaram-me incondicionalmente e tornaram-no possível. Por isso, a

vocês agradeço-vos tudo, mas mesmo tudo, não só pela educação que me deram, mas

também por todos os ensinamentos, por nunca me fazerem desistir dos meus objetivos

e por serem os melhores PAIS do MUNDO, com quem eu posso sempre contar. À minha

mãe, em especial, gostaria de agradecer todos os conselhos, por me ouvir nos bons e

maus momentos, por me apoiar incondicionalmente, por me aconselhar e me fazer

acreditar que também sou capaz! À minha irmã, Rita, nem sei por onde começar… Sei

que a nossa relação como irmãs é diferente, somos cúmplices uma da outra e não há

ninguém que me conheça tão bem como tu! És sem dúvida a melhor irmã que podia ter

tido e apesar das nossas birras, sei perfeitamente que não conseguíamos viver uma

sem a outra! Gostaria de te agradecer por todas as palavras, nos bons e maus

momentos, toda a ajuda durante estes anos! Foste a melhor parceira de estudo que

podia ter tido, sempre muito paciente com os meus momentos de contradição perante

as evidências científicas mais do que comprovadas! És sem dúvida uma pessoa muito

paciente comigo! Quero ainda agradecer-te por toda a tua disponibilidade para me

ajudares, para me ouvires a treinar as orais (inúmeras vezes!) e por estares sempre

presente quando eu mais preciso de ti! Vocês os três são sem dúvida as pessoas mais

importantes da minha vida e a quem agradeço por tudo aquilo que sou e tenho!

Gostaria ainda de agradecer aos restantes elementos da minha família, tios,

primos, pelo apoio incondicional, pela preocupação e por estarem sempre presentes em

todos os momentos.

Este ano foi sem dúvida alguma inesquecível. Não podia pedir mais para o meu

ano de Tese! Se há um ano me dissessem que ia ter um ano tão incrível como este não

ia acreditar! Levo comigo todos os momentos, todas as aprendizagens! Obrigada por

me mostrarem o quão fascinante é a CIÊNCIA, a INVESTIGAÇÃO e me fazerem

confirmar mais uma vez que isto é realmente o que eu quero SER!

IV FCUP/ICBAS Study of the cellular and molecular mechanism associated with neonatal susceptibility to bacterial infections


V

Resumo

A sepsis neonatal é responsável pela morte de aproximadamente 1.35 milhões

de bebés por ano.

Existem muitos microrganismos responsáveis pelo desenvolvimento de sepsis

bacteriana, sendo os mais relevantes em termos de incidência o Estreptococos do grupo

B (SBG), o Staphylococcus aureus, o Streptococcus pneumoniae, a Escherichia coli e

a Klebsiella pneumoniae. As infeções podem ser adquiridas antes, durante ou após o

nascimento, não existindo atualmente uma imunoterapia preventiva eficaz.

Estudos prévios do nosso grupo mostraram que, em murganhos, a

suscetibilidade neonatal à sepsis induzida por SGB estava associada a um aumento

precoce da produção de interleucina 10 (IL-10), uma citocina imunossupressora.

Identificamos a gliceraldeído-3-fosfato desidrogenase (GAPDH) de SGB na forma

extracelular como a responsável pela indução da produção desta citocina. A produção

de IL-10 a tempos precoces de infeção, impede o recrutamento de neutrófilos para os

locais de infeção, não ocorrendo por isso a eliminação da bactéria. Demonstramos ainda

que a neutralização da GAPDH bacteriana mediada por anticorpos era eficiente na

proteção de ratinhos recém-nascidos contra sepsis induzida por SGB.

Sendo a GAPDH bacteriana uma proteína muito conservada, a nossa hipótese

é que diferentes bactérias associadas com infeções neonatais utilizem a GAPDH como

forma de escape ao sistema imune do hospedeiro. Desta forma, decidimos avaliar se o

S. aureus e o S. pneumoniae usam a GAPDH como mecanismo de evasão ao sistema

imune do hospedeiro. Em paralelo, fomos estudar o mecanismo molecular e celular

associado com a suscetibilidade a infeções causadas por SGB, S. aureus e S.

pneumoniae.

Para confirmar a nossa hipótese, desenvolvemos uma vacina baseada em

péptidos (PNV1) que estão presentes na superfície da GAPDH de SGB, de S. aureus,

de S. pneumoniae e de outros microrganismos gram-negativos. Usamos o hidróxido de

alumínio (Alhydrogel 2%) como adjuvante. Esta vacina mostrou ser imunogénica nos

diferentes modelos animais testados (murganhos, ratos e coelhos). Purificamos as

imunoglobulinas (Ig) anti-PNV1 (PNV1-IgGs) de ratos imunizados com PNV1 e

imunizamos passivamente murganhos recém-nascidos antes da infeção com SGB, S.

aureus e S. pneumoniae. Como controlo foram utilizados IgG purificados de ratos

controlo.

Os resultados obtidos mostraram que a imunização passiva com PNV1-IgGs

conferiu proteção ao induzir um aumento significativo na sobrevivência dos animais

VI FCUP/ICBAS Study of the cellular and molecular mechanism associated with neonatal susceptibility to bacterial infections

infetados, para todas as bactérias em estudo, quando comparado com os murganhos

que receberam IgGs controlo.

Dados não publicados do nosso grupo tinham demonstrado que as células B

CD5+ (células B1) eram uma importante fonte de IL-10 induzida pela GAPDH bacteriana.

Além disso, o nosso grupo tinha mostrado que em murganhos recém-nascidos, knock-

out para o gene do TLR2 (TLR2-/-) e infetados com SGB não se observava a produção

de elevados níveis de IL-10 após a infeção e, consequentemente, eram mais resistentes

do que os murganhos controlos wild-type (WT). Para verificarmos se o TLR2 era um

recetor para a GAPDH de SGB, purificamos células B1 do baço de murganhos TLR2-/-

que foram estimulados in vitro com GAPDH bacteriana. Verificamos que células B1 de

murganhos TLR2-/- perdiam a sua capacidade para produzir IL-10 em resposta à GAPDH

bacteriana, sugerindo que o TLR2 poderá ser o recetor para a GAPDH bacteriana. Para

confirmar, infetamos murganhos recém-nascidos WT e TLR2-/- com S. aureus ou com S.

pneumoniae. Foi-nos possível observar que murganhos TLR2-/- apresentam um

aumento da sobrevivência após infeção com qualquer uma das bactérias, à semelhança

do que tinha sido observado com o SGB.

Em suma, os resultados apresentados neste estudo clarificam os mecanismos

responsáveis pelo aumento da suscetibilidade dos recém-nascidos contra diferentes

infeções bacterianas e avançam com uma nova vacina como possível candidata para a

prevenção de infeções neonatais, para as quais não existe nenhuma terapia atualmente

disponível.

Palavras-chave: infeções neonatais; SGB; Staphylococcus aureus; Streptococcus

pneumoniae; GAPDH; interleucina-10; PNV1; vacina


VII

Abstract

Neonatal sepsis is responsible for the death of approximately 1.35 million babies

per year.

The bacterial pathogens that are more frequently associated with the cases of

neonatal sepsis are group B Streptococcus (GBS), Staphylococcus aureus,

Streptococcus pneumoniae, Escherichia coli and Klebsiella pneumoniae. These

infections can be acquired before, during or after birth, and currently there is no effective

immunotherapy to prevent neonatal bacterial infections.

Previous studies from our group showed that, in mice, the neonatal susceptibility

to GBS-induced sepsis is related to an innate commitment of neonates to produce high

levels of interleukin-10 (IL-10), an immune-suppressive cytokine. We identified the

extracellular form of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the

responsible for the induction of IL-10 production. We also showed that GBS GAPDH

induces IL-10 production at early time points of infection, disabling neutrophil recruitment

into infected sites and bacterial clearance. Importantly, we demonstrated in mice that

antibody-mediated neutralization of bacterial GAPDH was very effective in protecting

newborns from GBS sepsis.

Being bacterial GAPDH a very conserved protein, our hypothesis is that different

bacteria associated with neonatal infections also use GAPDH as a form to escape from

host immune system. Thus, we aimed to evaluate if S. aureus and S. pneumoniae use

GAPDH as a mechanism to evade from host immune system. In parallel, we also focus

our attention on the comprehension of the molecular and cellular mechanisms

associated with neonatal susceptibility to infections caused by GBS, S. aureus and S.

pneumoniae.

To confirm our hypothesis, we developed a peptide-based vaccine (PNV1),

composed of surface-exposed peptides present in the GAPDH from GBS, S. aureus, S.

pneumoniae and other gram-negative microorganisms. Aluminium hydroxide

(Alhydrogel 2%) is the adjuvant used in PNV1. This vaccine showed to be immunogenic

in the different animal models tested (mice, rats and rabbits). Purified immunoglobulins

(Ig) from PNV1-immunized rats were used for passive immunizations in neonatal mice

before infection with GBS, S. aureus and S. pneumoniae. As controls we used IgG

purified from sham-immunized rats.

Importantly, we observed that passive immunization with PNV1 IgGs conferred a

significant increase in the survival of animals infected with all the referred bacteria, when

compared with mice that received control IgGs.

VIII FCUP/ICBAS Study of the cellular and molecular mechanism associated with neonatal susceptibility to bacterial infections

Previous unpublished data from our group identified CD5+ B cells (B1 cells) as

an important source of GAPDH-induced IL-10. Moreover, we also found that upon GBS

infection, TLR2 knockout (TLR2-/-) mice do not produce significant amounts of IL-10 and,

consequently, are more resistant than wild-type (WT) pups. In order to confirm if TLR2

was the GBS GAPDH receptor, we purified B1 cells from the spleen of TLR2-/- neonatal

mice and stimulated them in vitro with bacterial GAPDH. Interestingly, B1 cells from

TLR2-/- mice loss the ability to produce IL-10 in response to GAPDH, suggesting that

TLR2 is the cellular receptor for bacterial GAPDH. To confirm this, we infect TLR2-/-

neonatal mice with S. aureus and S. pneumoniae. We observe that TLR2-/- mice have an

increase of survival upon bacterial infection, as observed previously in GBS infection.

Altogether, the results presented in this study indicate that there is a common

virulence mechanism that is shared by different bacterial pathogens and it is responsible

for the increased susceptibility of neonates against infections caused by GBS, S. aureus

and S. pneumoniae. Moreover, with the data presented herein we suggest a new vaccine

candidate for the prevention of neonatal infections, a current unmet medical need.

Keywords: neonatal infections; GBS; Staphylococcus aureus; Streptococcus

pneumoniae; GAPDH; Interleukin-10; PNV1; vaccine


IX

X FCUP/ICBAS Study of the cellular and molecular mechanism associated with neonatal susceptibility to bacterial infections


IX

Index

Aknowledgments .............................................................................................. I

Resumo ............................................................................................................ V

Abstract .......................................................................................................... VII

Figure and Tables ........................................................................................... XI

Abbreviations ............................................................................................... XIII

Introduction ..................................................................................................... 1

1. Neonatal Sepsis ..................................................................................... 3

1.1 Overview .......................................................................................... 3

1.2 Epidemiology .................................................................................... 4

1.3 Disease ............................................................................................ 5

1.3.1 Early and Late Onset Sepsis ......................................................... 5

1.4 Most relevant Gram-positive pathogens in neonatal sepsis .............. 7

1.4.1 GBS .............................................................................................. 7

1.4.2 S. aureus ...................................................................................... 8

1.4.3 S. pneumoniae .............................................................................. 9

1.5 Diagnosis ........................................................................................ 10

1.6 Prophylaxis for neonatal infections ................................................. 11

1.6.1 Before delivery ............................................................................ 11

1.6.2 During Labour and Delivery ........................................................ 11

1.6.3 After delivery ............................................................................... 13

2. Neonatal Immune response ................................................................ 14

3. Protein Virulent Factors as New Targets ........................................... 17

3.1 GAPDH........................................................................................... 18

3.1.1 B1 cells are responsible for IL-10 production upon bGAPDH

stimulus………………………………………………………………………..19

Aim of the study ............................................................................................ 23

Material and Methods .................................................................................... 25

Bacterial Strains and Growth conditions ................................................. 27

Animals and Ethics statements ................................................................ 27

X FCUP/ICBAS Study of the cellular and molecular mechanism associated with neonatal susceptibility to bacterial infections

Preparation of Neonatal Vaccine .............................................................. 28

Neonatal mouse model of infection .......................................................... 28

Purification of anti-PNV1 IgGs .................................................................. 28

PNV1 IgG passive immunizations ............................................................. 28

Quantification of serum IgG ...................................................................... 29

Purification of the bacteria rGAPDH ......................................................... 29

Crude extracellular proteins (CEP) ........................................................... 30

Western Blot analysis ................................................................................ 30

Cell cultures ............................................................................................... 30

1. B1 cell purification .............................................................................. 30

2. IL-10 quantification ............................................................................. 31

Statistical analysis ..................................................................................... 31

Results ........................................................................................................... 33

PNV1 immunogenicity ............................................................................... 35

PNV1-elicited antibodies react with extracellular bacterial GAPDH ....... 36

TLR2 signalization is required for IL-10 production mediated by B1 cells

upon bacterial GAPDH ............................................................................... 39

TLR2 KO animals have an increased survival upon bacterial infection . 40

PNV1 as an efficient therapy against bacterial infection ........................ 42

Discussion ..................................................................................................... 45

Future works .................................................................................................. 53

Conclusion ..................................................................................................... 57

Bibliography .................................................................................................. 61


XI

Figure and Tables

Figure 1: Causes of death among children under 5 years. ........................................... 4

Figure 2: Stages of neonatal infection. ......................................................................... 6

Figure 3: IAP. ............................................................................................................. 12

Figure 4: Antibiotic-resistant bacteria. ........................................................................ 14

Figure 5: GBS GAPDH. ............................................................................................. 18

Figure 6: Differences in IL-10 production between different population of cells. ......... 20

Figure 7: IL-10 production mediated by B cell populations. ........................................ 21

Figure 8: Serum anti-GAPDH IgG antibodies in mice immunized with PNV1. ............ 35

Figure 9: Serum anti-PNV1 IgG antibodies titers in rats and rabbits immunized with

PNV1. ......................................................................................................................... 36

Figure 10: Western Blot analysis of Gram-positive, Gram-negative and human rGAPDH.

................................................................................................................................... 37

Figure 11: Serum anti-Human GAPDH IgG in mice immunized with PNV1. ............... 38

Figure 12: PNV1-elicited antibodies were able to recognize bacterial GAPDH in the

bacterial CEP. ............................................................................................................. 38

Figure 13: GAPDH-mediated IL-10 production in mice B1 lymphocytes is blocked when

TLR2 signaling is inhibited. ......................................................................................... 40

Figure 14: TLR2-/- newborns mice have an increased of survival upon bacterial infection.

................................................................................................................................... 41

Figure 15: PNV1 immunization protects newborn mice against bacterial infection. .... 43

Figure 16: PNV1-elicied antibodies protection against neonatal bacterial infections... 52

Table 1: List of bacteria and respective strains used during the experiments. ............ 27

XII FCUP/ICBAS Study of the cellular and molecular mechanism associated with neonatal susceptibility to bacterial infections


XIII

Abbreviations

2-ME – 2-mercaptoetanol

AAP – American Academy of Pediatrics

ACOG – American College of Obstetricians and Prevention

AP – alkaline phosphatase

APCs – antigen-presenting cells

APPs – antimicrobial proteins and peptides

BCIP – 5-bromo-4-chloro-3`-indolyphosphate p-toluidine salt

bGAPDH – bacterial GAPDH

BPI – bactericidal/permeability-increasing protein

BSA – bovine serum albumin

CD – cluster of differentiation

CDC – Centers for Disease control and Prevention

CEP – crude extracellular products

CNS – central nervous system

CoNS – coagulase negative staphylococci

CPS – capsular polysaccharide

CR – complement receptor

CRP – C-reactive protein

cRPMI – complete RPMI

CSF – cerebrospinal fluid

E. coli – Escherichia coli

EOD – early-onset disease

EOS – early-onset sepsis

FBS – fetal bovine serum

FLDC – dendritic cells

FLM – macrophages

FQRP - fluoroquinolone-resistant Pseudomonas aeruginosa

GAPDH – glyceraldehyde 3-phosphate dehydrogenase

GBS – Group B Streptococcus

HIV – human immunodeficiency virus

HRP – Horseradish peroxidase

IAP – intrapartum antibiotic prophylaxis

IL – interleukin

Ig - immunoglobulin

XIV FCUP/ICBAS Study of the cellular and molecular mechanism associated with neonatal susceptibility to bacterial infections

I.P. – intraperitoneally

IPTG – isoprophyl-β-D-thiogalactopyranoside

i3S – Instituto de Investigação e Inovação em Saúde

K. pneumoniae – Klebsiella pneumoniae

LB – Luria-Bertani

LBP – lipopolysaccharide-binding protein

LPS – lipopolysaccharide

LOD – late onset disease

LOS – late onset sepsis

MD - myeloid differentiation

MNC – spleen mononuclear cells

MRSA – Methicillin resistant Staphylococcus aureus

NBT – nitro-blue tetrazolium chloride

NICU – neonatal intensive care unit

NT – nontypeable

PAMPs – pathogen-associated molecular patterns

PLA2 – group II phospholipase

PCR – polymerase chain reaction

PCV – pneumococcal conjugate vaccine

PepO – endopeptidase O

PMNC – neutrophils from peripheral blood

PPV – pneumococcal polysaccharide vaccine

rGAPDH – recombinant GAPDH

rPepO – recombinant endopeptidase O

ROS – reactive oxygen species

S.C. - subcutaneously

S. aureus – Staphylococcus aureus

S. pneumoniae – Streptococcus pneumoniae

TH – Todd-Hewitt

TH – T helper

TLR – toll-like receptor

TLR2-/- – TLR2 knockout

TMB – 3,3`,5,5`-tetramethylbenzidine

TNF-α – tumour necrosis factor alpha

Treg – regulatory T cells

VLBW – very-low-birthweight

VIP – virulent associated immunomodulatory protein


XV

VRE – vancomycin-resistant Enterococcus

WB – western blot

WHO – World Health Organization

WT – wild-type

XVI FCUP/ICBAS Study of the cellular and molecular mechanism associated with neonatal susceptibility to bacterial infections


1

Introduction

Chapter 1

2 FCUP/ICBAS Study of the cellular and molecular mechanism associated with neonatal susceptibility to bacterial infections


3

1. Neonatal Sepsis

1.1 Overview

Neonatal sepsis or septicaemia is defined as a systemic inflammatory response

caused by an infection and it is characterized by an acute circulatory failure that can

rapidly develop into septic shock [1].

In the pre-antibiotic era, neonatal sepsis was usually fatal. However, with the

introduction of antibiotics, the fatality range decreased for values between 5% and 60%

[1]. Currently and as expected the highest incidence is reported in low-income countries

[2, 3]. There are several factors that can contribute to the occurrence of bacterial

infections in the neonates, such as the lack of neonatal care, preterm birth, low

birthweight and unhygienic or unsafe delivery practices [3]. The infection can be acquired

before, during or after birth [1].

The agent more frequently associated with neonatal bacterial sepsis is

Streptococcus agalactiae, also known as Group B streptococcus (GBS), a Gram-positive

bacterium. However, other Gram-positive and Gram-negative bacteria are also frequent

agents of severe neonatal infections, such as Staphylococcus aureus (S. aureus),

Streptococcus pneumoniae (S. pneumoniae), Escherichia coli (E. coli) and Klebsiella

spp [4, 5]. GBS, Staphylococcus spp. and E. coli, in combination, contribute to

approximately 58% of neonatal infections [6].

Newborns are particularly susceptible to infections due to their immature immune

system. The neonatal period of life corresponds to a developmental stage in which

responses are dependent on the conditions of antigen exposure [7]. Neonates just rely

to their innate immune system to respond to bacterial infections [8]. Newborns have an

impaired neutrophil recruitment, and phagocytic capacity, especially preterm infants [9,

10]. In addition, they have no ability to produce high levels of immunoglobulins (Ig) G

(IgG) in response to infection and tend to produce high levels of the immunosuppressive

cytokine – interleukin (IL) 10 [11].

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme

and previous work from our group showed that GBS GAPDH is a virulence-associated

immunomodulatory protein (VIP) [11]. During infection there is a compromise in the

neutrophil recruitment due to a high production of IL-10 mediated by toll-like receptor

(TLR) 2 [12]. Moreover, another study demonstrated that maternal immunization with

GBS recombinant GAPDH (rGAPDH) leads to a decrease of IL-10 production and

consequently protects neonates against GBS infection [13]. Taking into account the role


of GAPDH in GBS virulence and the highest conservation of this protein between

different pathogens, the use of a vaccine based on the structure of this protein may be

an effective vehicle to prevent neonatal infections.

1.2 Epidemiology

Neonatal sepsis is an important cause of mortality and morbidity [14]. The World

Health Organization (WHO) estimates that from the approximately 4 millions of the

neonatal deaths that occur every year, 1 million are due to neonatal sepsis and 42%

occur in the first month of life [1, 3]. There is also a high morbidity amongst the survivors

of bacterial infections, with 30-50% of surviving neonates suffering from severe sequels

[15]. Recent data from 2015 (Figure 1) demonstrates that approximately 7% of all

neonatal deaths among children under five years are due to the development of sepsis.

The medium rate mortality during the neonatal period is thirty times higher than

in the post-neonatal period. Moreover, it should be taken in account that mortality in the

first twenty-four hours of life is higher and that approximately three-quarters of neonatal

deaths happen in the first week of life [3].

Figure 1: Causes of death among children under 5 years. Representative diagram of the most important causes of

death among children under five years, in 2015, provided by WHO.

(http://www.who.int/gho/child_health/mortality/causes/en/)

Neonatal meningitis has a significant impact in neonatal morbidity and mortality,

with neonates presenting a rate of meningitis higher than other groups [16, 17]. The

incidence in developed countries had declined in the last years, from 50% in the 1971s

to less than 10% in 1997 [18]. Nevertheless, the morbidity of neonatal meningitis remains

http://www.who.int/gho/child_health/mortality/causes/en/


5

stable, with an incidence of 20-58%, and the survivors can develop severe sequels. In

developing countries, the mortality due to meningitis has a higher rate, with an incidence

between 40-58% [19].

In preterm infants (<28 weeks of gestation) there was an increase in sepsis

hospitalization in the early to mid-1990s, but no significant changes occurred thereafter

[6].

The most important organisms in neonatal sepsis are GBS, S. aureus, S.

pneumoniae, E. coli, Klebsiella spp. and Pseudomonas aeruginosa [4, 5]. GBS is the

most important agent in developed countries, whereas in developing countries the causal

organism are frequently Gram-negative agents such as Klebsiella spp., and E. coli [20].

1.3 Disease

1.3.1 Early and Late Onset Sepsis

In terms of neonatal infections is important to consider the differences between

Early-Onset Sepsis (EOS) and Late-Onset Sepsis (LOS). In the first case, the symptoms

occur in the first seven days (<7 days) of life. Usually, EOS is the result of a vertical

transmission of the bacteria from the mother to the newborn, before or during birth [14,

21]. On the other hand, LOS symptoms occur after the first week of life and until the 120th

day [14, 22]. This type of infection can be acquired vertically or horizontally [14, 21], so

it can be transmitted at delivery or as a result of a nosocomial infection. Very-low-

birthweight (VLBW, <1500g birth weight) infants hospitalized in the neonatal intensive

care unit (NICUs) are especially susceptible to LOS due to their undeveloped immune

system and extended time of hospitalization [22] .

The incidence of EOS in United States is approximately 0.77 to 1 per 1000 live

births. Black preterm infants (<37 weeks of gestation) had a highest incidence of

infections – 5.14 cases/1000 live births – than Non-Black preterm infants (≥37 weeks of

gestation). The same occurs when comparing the incidence in Black term infants (≥37

weeks of gestation) and non-black term infants [23].

Transmission of the bacteria into the newborns can occur in several ways: before,

during or after birth. Infections before birth result from the ascension of commensal

bacteria from mother’s genital tract into the amniotic fluid. The infections occurring during

labour are mainly due to the aspiration of pathogens that colonize the mother genital

tract mucous. Infections occurring after birth are usually nosocomial infection [1].

As shown in the Figure 2, the first focus of neonatal infection is the lung. After

that, the bacteria can have access to the bloodstream and, consequently, to other


anatomical places, causing bacteraemia and sepsis syndrome. Finally, bacteria can gain

access into the brain and central nervous system (CNS), resulting in the development of

meningitis [24].

Figure 2: Stages of neonatal infection. Schematic representation of the steps of neonatal infection, indicating the

progressive focus of infection, which result in the development of meningitis. Adapted from [24].

When VLBW infants survive neonatal sepsis, they have a higher risk of

developing morbidities, namely periventricular leukomalacia, neurological impairment

and bronchopulmonary dysplasia [22, 25].

Risk factors for EOS can include maternal or infant factors. The first include, for

example, prolonged rupture of the membrane (>18h), intrapartum fever, vaginal

colonization with GBS at delivery or at 28 or 36 weeks of gestation, chorioamnionitis [26-

28], history of previous infants with infection [29] and low levels of type specific IgG

antibodies [28]. It is generally accepted that transplacental transfer of maternal derived

antibodies against GBS capsular antigens protects neonates against these infections

[28, 30]. Gestational age and exposure to antenatal antibiotic are the most important

factors to take in account in LOS [22].

GBS is the most frequent agent of EOS, but E. coli is the most common cause of

mortality (24%). These two microorganisms account for approximately 70% of EOS

infections [14]. GBS is most incident bacteria among term infants and E. coli among

preterm infants [23]. Between 1997 and 2010 a study with VLBW showed that EOS

mortality rate was similar for Gram-positive and Gram-negative bacteria: 24,7% and

28.0%, respectively [22].

During the same period, results had shown that LOS is caused, essentially, by

Gram-positive microorganisms (61,4%), followed by Gram-negative (26,2%). The most

important agents are Coagulase negative staphylococci (CoNS), S. aureus and other

Gram-positive cocci [22].


7

1.4 Most relevant Gram-positive pathogens in neonatal sepsis

1.4.1 GBS

GBS was first described as a cause of bovine mastitis [31]. GBS is a β-hemolytic

and Gram-positive bacterium and is the leading agent of neonatal infections, which can

cause sepsis, meningitis, pneumonia and also death [32]. It can also cause invasive

disease in pregnant women, and nonpregnant adults with underlying medical conditions

[33]. GBS is a commensal bacterium that is present in the human gastrointestinal flora

and colonizes the vagina in 10-50% of women, and 15-35% of pregnant women [34, 35].

Levels of colonization are similar in women from low-income or high-income countries

[32].

Clinical syndromes of GBS infection in newborns are sepsis, pneumonia,

meningitis, osteomyelitis, septic arthritis and, in a few percentage, endocarditis and

epiglottitis [34, 36].

GBS capsular polysaccharide (CPS) was recognized as an important virulent

factor with antiphagocytic function, and nowadays ten different serotypes are identified

(Ia, Ib, II-IX) [31]. Nontypeable (NT) strains of GBS are increasingly being recognised as

a frequent cause of severe infections [37]. As mentioned above, this type of infections

can be divided in EOS and LOS. Early onset disease (EOD) occurs in 60-70% of cases

[31] and the most important serotypes responsible for this form of disease are: Ia, II, III

and V [38]. Moreover, a study has shown that serotypes III, Ia and V account for 83% of

all EOD cases [39]. The pathogenesis of LOS is less understood, but it can be acquired

during hospital stay [40], from community sources or perinatally [31, 39]. The most

relevant serotype is serotype III and a study has shown that up to 50% of cases presents

with meningitis [39].

GBS infections are also reported in non-pregnant adults, mainly in adults with

more than 65 years [41]. The incidence is higher in adults with underlying medical

conditions such as diabetes, cirrhosis, neurogenic bladder, decubitus ulcer, breast

cancer, history of stroke and infection with the human immunodeficiency virus (HIV) [42,

43]. However, diabetes appear to be the most important condition, with 20-25% of

nonpregnant adults with this disorder presenting GBS infection [44].

In adults, the disease manifests as bone, joint, skin and soft tissue infections,

pneumonia, urosepsis, meningitis, endocarditis, infections in the intravascular catheters

or recurrent invasive GBS infection [44]. Trivalle et al [45] described the three most

important clinical diagnoses in older patients: urinary tract infection (39%), skin infection

(33%) and pneumonia (24%).


Rebeca Lancefield proposed in 1930 that protection against GBS infection could

be achieved using CPS-specific polyclonal rabbit serum [46]. Clinical trials with a vaccine

prepared with purified CPS types Ia, Ib, II, III showed that this treatment induced

functionally active CPS-specific IgG [47], and a bivalent vaccine demonstrated reactivity

and tolerance by healthy humans [48]. Nevertheless, the highest problem of this type of

vaccines is that it does not confer protection against other serotypes that are also

prevalent in different regions of the world [49].

1.4.2 S. aureus

Staphylococcus spp. are the second leading cause of LOD in NICUs patients

[50]. S. aureus is responsible for considerable morbidity and mortality. One of the most

significant obstacle to the treatment of S. aureus infections is the increase in the

incidence of antimicrobial-resistant strains, namely resistance to methicillin. The first

case was reported in 1961 [51] and after that Methicillin Resistant Staphylococcus

aureus (MRSA) infections emerged, particularly in the NICUs [52]. MRSA is resistant to

first line antimicrobial therapies and, as expected, infections with this pathogen are

associated with higher morbidity and increased medical costs [51]. Vancomycin is used

in the treatment of MRSA and multi-drug-resistant S. aureus infections, but resistant

strains to this antibiotic have already been reported [53].

It is also important to consider that VLBW have increased risk of infection due to

their immature immune system, exposure to invasive devices, procedures, and

prolonged hospital stays [50].

The virulence of Staphylococcus spp. strains increases due to their capacity to

form biofilms, with studies demonstrating that in CoNS infections, biofilms are important

in the development of nosocomial bacteremia [54]. Biofilm-formation occur in many

medical devices and catheters [54, 55], and is a characteristic that favors the survival of

Staphylococcus spp. in the tissues and blood, and increase their resistance to the

antibiotic therapies available [54].

Since there is no treatment available for S. aureus infections, it is important to

find a safe preventive measure to reduce this type of infections, particularly in neonates.

The research for new therapies led to the discovery of new antibiotics against S. aureus

infections, nine of them are in clinical trial. Also, other therapeutic measures have been

suggested, such as antibodies against staphylococcal toxins (e.g. alpha-hemolysin),

against staphylococcal surface proteins (e.g. protein A) or staphylococcal non-protein

antigens (e.g. CPS) [56].


9

1.4.3 S. pneumoniae

S. pneumoniae is a Gram-positive encapsulated diplococcus [57]. It can cause

invasive or non-invasive disease, mainly in the paediatric population, but also elderly and

immunocompromised individuals [58]. Pneumococcal pneumoniae is considered one of

the most important causes of childhood and adult mortality worldwide. WHO estimates

that 1.6 millions of deaths are caused by this agent annually and that 0.7-1 millions

children under the age of five die annually due to pneumococcal infection, mainly in low-

income countries [57]. Intrapartum colonization, histological chorioamnionitis and

maternal bacteraemia due to S. pneumoniae is rare, but this type of infection constitutes

an important risk factor for the development of EOS [59]. Moreover, when this type of

infection occurs, it has serious implications for both the mother and the newborn [60].

Previous studies demonstrated that invasive S. pneumoniae disease in neonates is

associated with maternal colonization, prematurity, prolonged rupture of membrane,

early-onset pneumoniae presentation and high mortality (50%) [61]. GBS and S.

pneumoniae sepsis are identical, but the latter appears to be more virulent and of marked

severity, which leads to a higher neonatal mortality [60].

There are currently 93 different serotypes of S. pneumoniae identified [62].

Nevertheless, there are some serotypes that are more associated with invasive

pneumococcal disease than others. WHO estimates that 80% of all invasive

pneumococcal disease were caused by 20 of the 93 serotypes [57].

One of the most important problems associated with this type of infections is the

pneumococcal resistance to antimicrobials such as the penicillins, cefalosporins,

macrolides and fluoroquinolones. This emphasizes the need for a preventive measure

through immunization [57].

Actually there are two pneumococcal vaccines available: pneumococcal

polysaccharide vaccine (PPV) and pneumococcal conjugate vaccine (PCV). The first

one includes 23 capsular polysaccharides and is used in elderly and in adults that are at

high risk of pneumococcal disease. PCV is constituted by capsular polysaccharides of

13 different serotypes conjugated with a carrier protein (Prevnar 13). It is licenced for

use in children aged less than five years, but is immunogenic in all age groups [57].

Nevertheless, the formulation of the vaccine is based on serotypes that most frequently

cause invasive disease, which can vary between different countries [58]. Thus, this

formulation cannot be effective in all countries, so it is essential to look for an effective

method to prevent this type of infections.


1.5 Diagnosis

The diagnosis of neonatal sepsis is problematic, because the clinical signals can

be confused with other life-threatening diseases [63, 64]. The identification of the

microorganisms responsible for neonatal sepsis is also very difficult, so actually there

are a range of techniques to identify the pathogenic organisms. However, it is important

to take in consideration that in some situations the identification is difficult due to the

administration of antibiotics to the mother, antepartum or intrapartum, which leads to a

reduction of bacterial load. Moreover, only a small amount of blood can be collected from

newborns [1, 65].

The blood culture methods have been long considered the standard

microbiological diagnosis. Nevertheless, this method has the disadvantage of requiring

up to 48h [66] and has a very low sensitivity [67]. For example, the diagnosis of GBS

infection is done using swabs (combination of swab of both vaginal and rectum is

preferable to other sites), which are inoculated in selective broth medium – Todd-Hewitt

(TH) broth supplemented [34]. The use of supplements aims to suppress the growth of

Gram-negative bacteria [68]. Results showed that when nonselective medium was used,

a high number of false-negatives results occurred (58,9%) [69]. The selective medium

containing the samples is incubated during 18-24h. After this period a subculture is made

to a sheep blood agar plate for 18-24h. If GBS is suspected, an inspection and

identification is made (beta-hemolytic or nonhemolytic, calatase-negative organisms or

Gram-positive). If GBS is not identified at the end of the incubation period, then the plate

is reincubated at 48h in order to identify suspicious organisms [34].

Another technique to detect pathogenic organisms include antigen detection

techniques, polymerase chain reaction (PCR) and biological markers [1, 67]. The first

one, allows rapid detection and identification of the microorganism. An example is the

latex agglutination assay, which consist in the agglutination by bacterial cell wall antigens

of antibody-coated latex particles. However, this type of test only allows for the detection

of specific organisms and is associated with a high ratio of false positive and false

negative results [1]. New urinary and cerebrospinal fluid (CSF) antigen test for the

detection of Pneumococcus infection demonstrated high sensitivity and sensibility in

samples of CSF with culture confirmed meningitis [70]. PCR has the advantage of

allowing the detection of bacteria even when the concentration is low [1]. Biological

markers are human blood components that increase during the response to infection.

One of the most common is the C-reactive protein (CRP) [1]. However, to measure this

protein, 12-24 hours are necessary. Other options have been studied, such as IL-6, IL-

8, tumour necrosis factor alpha (TNF- α) and procalcitonin detection [71].


11

1.6 Prophylaxis for neonatal infections

1.6.1 Before delivery

Before delivery the most important method to protect neonates from bacterial

infections is the maternal immunization that can provide neonates with appropriate levels

of IgG to specific antigens that are passively transferred across the placenta [72, 73].

Maternal vaccination has been suggested as a good alternative to prevent

neonatal infections. For example, a GBS type III CPS-tetanus conjugate vaccine

administered in the third trimester of pregnancy showed to be efficient in the prevention

of maternal, neonatal and young infant GBS disease [74]. Additionally, a modelling study

estimated that vaccination with an efficient GBS vaccine could prevent 60-70% of GBS

infections and 4% of preterm births, in the United States [75]. Encouraging results also

came from the immunization with PPV vaccine, demonstrating that this vaccine induces

an immunogenic response, that results in the production of antibodies that can be

transferred to the neonate via cord blood [76].

1.6.2 During Labour and Delivery

During labour and delivery there are evidences that clean delivery, handwashing

during delivery and maternal handwashing with soap and water or antiseptic, reduce the

neonatal mortality due to pneumonia, meningitis and sepsis [77]. Moreover, the use of

chlorhexidine appears to be beneficial, reducing neonatal and maternal mortality and

morbidity [78].

For GBS infections the use of intrapartum antibiotic prophylaxis (IAP) reduced

significantly the incidence of neonatal infections. From 1992 to 2002, several changes

have occurred in order to improve the best prophylaxis. The first measure of prophylaxis

was implemented in 1992 by the America Academy of Pediatrics (AAP) and consists in

a culture-based screening at 26 to 28 weeks of gestation. In 1996, the Centers for

Disease Control and Prevention (CDC), the American College of Obstetricians and

Gynecologists (ACOG) and the AAP recommended the implementation of two preventive

strategies. The first corresponded to the culture-based strategy, advising the

administration of antibiotics to women with prenatal rectal and vaginal screening positive

for GBS at 35 to 37 weeks of gestation. It is further indicated for women with previous

history of infants with GBS disease, membrane rupture for 18h or more, GBS bacteriuria

during pregnancy, women who deliver at less than 37 weeks, and women that present

intrapartum fever (≥38⁰C). The second strategy correspond to a risk-based strategy,

when IAP is applied to women that present one or more of the risk factors mentioned

above, but screening cultures are not applied. In 2002, adjustments to these measures


were made, and the first option was considered to be more effective than the second

one [79]. In the Figure 3 is summarized the current algorithms for the administration of

IAP issued in CDC guidelines.

Figure 3: IAP. IAP for GBS infections applied to pregnant women, based on vaginal and rectal GBS screening cultures

at 35-37 weeks of gestation. Adapted from [79].

IAP contributed for a decreased incidence of neonatal infection caused by GBS.

However, between 1999 and 2002, the ratio remained stable. After the adoption of

prophylactic measures in 2002, there was a further marked reduction in the cases of

EOD, and compared to the interval between 2000 and 2001, in 2004 there was a

decrease of 31%. However, the incidence of late onset disease (LOD) remained

unchanged, despite the widespread use of IAP [79]. In fact, in 2003 the rate of LOD

exceeded the rate of EOD [80]. Another consequence of this measures was the increase

of Gram-negative neonatal sepsis, particularly caused by E. coli. In a study performed

between 1991 and 1993, and between 1998 and 2000 (when IAP measures were

applied), it was demonstrated that the majority of neonatal infections between 1991 and

1993 were caused by Gram-positive organisms, more specifically by GBS. However,

between 1998 and 2000, the rate of GBS infections declined when compared to the first

period – 5.9 to 1.7 per 1000 live births. On the other hand, the rate of Gram-negative

infections increased and E. coli appeared as the most important organism of neonatal

infections (infections increased from 3.2 to 6.8 per 1000 live births). In the second period,


13

60.7% of neonatal infections were caused by Gram-positive organisms, whereas the E.

coli infections corresponded to 44% [81].

1.6.3 After delivery

Neonatal immunization has been recognized as an important strategy to reduce

neonatal infections. Nevertheless, the response suffers variations according to the

antigen [82]. Additionally, maternal antibodies also interfere with neonatal response to

vaccines, as observed for example in tetanus and hepatite A immunization [83].

Currently, the treatment of neonatal infections is made using parenteral

(intravenous or intramuscular) antibiotics (penicillin/ampicillin and gentamicin, or third-

generation cephalosporins alone – e.g. ceftriaxone or cefotaxime) for 10-14 days [1].

Although the first-line of treatment continued to be ampicillin and gentamicin, ampicillin

has the disadvantage that it needs to be administered more frequently [84].

Nevertheless, in general, ampicillin is preferred to penicillin. This results from the fact

that ampicillin has a higher activity against Gram-negative pathogens, when compared

to penicillin. However, the last one has higher activity against group A and B streptococci

and pneumococci and it is preferred in the treatment of meningococcal infections [84].

Nevertheless, the susceptibility of neonatal pathogens to this therapy vary:

Streptococcus spp. are usually susceptible [84] but Staphylococcus spp. can be highly

resistant. A study demonstrated that among Klebsiella spp., almost all were resistant to

ampicillin, 45% were resistant to cotrimoxazole, 66% to third generation cephalosporins

and 60% to gentamicin. E. coli presents lesser resistance to gentamicin (13%), but

resistance to ampicillin and third generation cephalosporins have been reported [5, 85].

The last option is the use of chloramphenicol but it is contraindicated in premature or low

birth weight infants due to it’s toxicity and secondary effects [84].

Another alternative of treatment is the oral antibiotic therapy instead of parenteral

antibiotic therapy. This must be considered in developing countries with limited health

system capacity. The most promising oral agents are the second generation

cephalosporins, cotrimoxazole, ciprofloxacin and amoxicillin. Nevertheless, the use of

cotrimoxazole has led to an increase of resistance to this therapy among pneumococcal

isolates. On the other hand, the oral administration of amoxicillin is effective against S.

pneumoniae, but it has no anti-Staphylococcus coverage and Gram-negative bacillus

resistance is emerging [86].

However, it is important to note that when such therapy is used, in most of the

cases, the infection is already in a very advanced state. As a consequence, a higher

number of newborns that survive present severe sequels. This effect and the constant

increase of bacterial resistance to antibiotics (Figure 4) [87] leads to the urgent need of


a human neonatal vaccine that could be efficient against various agents responsible for

neonatal bacterial sepsis.

Figure 4: Antibiotic-resistant bacteria. Representation of the increase in the percentage of antibiotic-resistant clinical

isolates of MRSA, Vancomycin-resistant Enterococcus (VRE) and fluoroquinolone-resistant Pseudomonas aeruginosa

(FQRP) between 1980-2005 [87].

Due to the immature neonatal immune system, the maternal vaccination appears

to be the best option, taking in consideration that in the first months of life neonate

protection against infectious diseases is highly dependent of passive immunization. This

type of immunization is dependent of specific IgG antibodies, that are transferred trough

the placenta, from the mother to the foetus [72, 73].

2. Neonatal Immune response

The fetal and neonatal immune system is associated with three physiological

demands: protection against bacterial and viral pathogens [88], prevention of the

potentially harmful effect of pro-inflammatory/T helper (TH) 1-cell-polarizing responses

which are responsible for recurrent aborts [89], and mediation of the transition from the

intra-uterine sterile environment to the antigen-rich environment of the outside world (for

example the first colonization of the skin [90] and intestinal tract by microorganisms [91]).

Newborns have an immature immune system which leads to an incomplete

immune response to the infections that is initially dependent of the innate immunity

because the adaptive immune system is not fully developed [8].


15

Human newborns present impaired TH1 cells responses, an excess of cytokines

(IL-6, IL-10 and IL-23) produced by monocytes and antigen-presenting cells (APCs) in

response to stimulus [92-94] and low levels of immunoglobulin, except IgG to specific

maternal antigens transferred passively across the placenta during the last trimester of

pregnancy [72, 73]. The diminished TH1 cells may be due to the predominance of a TH17-

like pattern combined with considerable IL-10 production [95].

After birth a maturation of the immune system occurs. For that the exposure to

environmental microbial products is important. The repeated exposure to the same

products accelerates maturation [96] by diminishing TH2 cell polarization and/or

enhancing TH1-cell polarization [97].

The uterine cavity presents a sterile environment, which is essential to protect the

fetus from infections. For example, it is important to take in account that cervical plug,

which separate the vagina (that normally has multiple microorganisms) from the normally

sterile intra-uterine environment, contains antimicrobial proteins and peptides (APPs),

including α-defensins, lysozyme and lactoferrin [98]. The amniotic fluid also contains

APPs, such as lactoferrin [99], histones (H2A and H2B) [100], defensins, and also

soluble cluster of differentiation (CD) 14 and lipopolysaccharide (LPS)-binding protein

(LBP). The last one binds CD14 and mediates the host response to Gram-negative

infections [101]. In the pre-term labour, an increase of group II phospholipase A (PLA2)

[102], an important enzyme against Gram-positive agents, occurs [103]. Moreover, the

APPs listed above confer protection against Gram-positive, but also against Gram-

negative agents [82].

After birth there is a fast colonization of the skin and gut. The neonatal skin is

particularly susceptible to infection. For example, soon after birth, approximately 50% of

the newborns develop Erythema toxicum neonatorum, which results from the reaction of

neonatal skin to commensal flora, in particular Gram-positive staphylococci [90]. So, a

series of defences were developed, in order to protect the newborn. One of the most

important is the vernix caseosa, a waxy coating secreted by sebaceous glands that

contains, for example, APPs and free fatty acids with important antimicrobial properties.

An in vitro study demonstrated that vernix caseosa is important for the host response

against bacterial and fungi infections, including E. coli, GBS and Candida albicans

infections [104]. Moreover, epithelial cells from the skin of embryonic and newborn mice,

as well as human newborn foreskin, express more cathelicidin and β-defensin

antimicrobial peptides than adults, which have important antimicrobial activity against

GBS and other bacteria [105].

In relation to the newborn intestinal tract, it is well known that upon delivery there

is a rapid transition to primary colonization, which is essential for the development of the


immune system of neonates [82]. Studies demonstrated that at 18-21 weeks of gestation

enterocytes in the small intestine of the human fetus express basolateral TLR2 and TLR4

[106]. On the other hand, fetal intestinal villous and crypt epithelial cells express TLR4

and myeloid differentiation (MD) protein 2 (MD2), which participate in the response to

LPS and Gram-negative bacteria [107]. Moreover, Paneth cells in the small intestine are

rich in APPs, which contributes to the clearance of bacteria such as E.coli [108].

Neonatal immunity is also modified by breastfeeding. Breast milk contains

immunological factors, including innate immune molecules, such as lactoferrin, APPs

and lysozyme [82, 109]. Furthermore, tolerance mechanisms are also triggered in order

to guarantee an inhibition of signalling involving TLR2 and TLR4 activation. The reduced

reactivity involving TLR2 facilitates the establishment of beneficial Gram-positive

bifidobacteria intestinal flora [82].

The respiratory tract is an important route of exposure to environmental antigens

and adjuvants and consequently has an important role in modulating the balance of

immune maturation. It is known that exposure to TLR agonists early in life accelerates

the maturation of TH1 cells and protects from atopic diseases and allergies [97].

However, at birth occurs an abundant expression of CD4+CD25+ regulatory T cells (TReg)

[110], that inhibit the immune responses of TH1 cells thereby maintaining peripheral T-

cell tolerance. TLR response can reverse the TReg cell function and, consequently,

enhance TH1-cell type response. However, further studies are needed to clarify the role

of TLR expression and effects of TReg cells [82].

Newborn mammals have a limited number of quiescent monocytes and

granulocytes. Additionally, the susceptibility to bacterial infections increases due to low

neutrophil storage pool and impaired capacity to migrate to the site of infection. As

known, neutrophils are essential cells in the innate immune response to bacterial

infections since they have cytoplasmic granules with microbicidal proteins and peptides.

For example, the presence of bactericidal/permeability-increasing protein (BPI) is

important in the protection against Gram-negative bacteria due to their capacity to bind

to bacterial LPS [111]. It was found that neonatal neutrophils show an impairment in

several aspects, including chemotaxis, rolling adhesion, transmigration and lamellipodia

formation [112, 113]. As we would expect, this situation results in impaired neutrophil

recruitment to the site of inflammation, which may be due to a decrease of integrins (one

of the most important is the complement receptor (CR) 3) and selectins (mainly L-

selectin) [114-116] expression, or even to an increase of the production of IL-6 (cytokine

that inhibits neutrophil migration into the inflammatory site) by the monocyte cells and

APCs [117]. Also noteworthy, neonatal neutrophils exhibit reduced expression of APPs

and BPI, but a normal expression of defensin peptides. As expected, these changes are


17

reflected in the function of neutrophils, and studies have shown that reduced expression

of BPI correlates with a reduction in neutrophil activity against Gram-negative bacterial

pathogens [111].

Another aspect to consider is the expression of TLRs, since they are essential for

the recognition of bacterial structures called pathogen-associated molecular patterns

(PAMPs) [118]. For example, bacterial lipoproteins are recognized by TLR2 and LPS by

TLR4 [119]. Cord-blood monocytes of preterm humans have decreased basal

expression of TLR4, but a normal expression of TLR2, compared with adult peripheral-

blood monocytes [82, 120].

3. Protein Virulent Factors as New Targets

Nowadays, the vaccination appears to be the most effective treatment for

neonatal infections, because it can protect adults, but also newborns, without the

problems associated with antibiotic use [121, 122]. The vaccination program must target

pregnant women for immunization, in order for a passive transference of antibodies

across the placenta to occur [72, 73].

The investigators believe that the most effective strategy against neonatal

bacterial infection should be based on conserved antigenic proteins [32], to avoid the

selection of mutants that escape immune recognition. Moreover, the vaccine should be

directed to important antigens for the growth and/or virulence of the pathogen [123].

As mentioned above, one of the vaccines proposed for GBS infections is based

on the CPS, being a bivalent vaccine used in clinical trials [48]. However, this formulation

has limitations due to the distinct distribution of serotypes in different regions [49]. Based

on this, other vaccine candidates have been suggested for GBS infections, such as

protein-based vaccines. The most relevant targeted proteins include Rib, Sib and C5a

peptidase, all of which correspond to surface proteins. The first one is a surface protein

with homology with the α component of protein C. Being the C protein also a candidate

for GBS vaccine, the combination with Rib and α component was suggested. Sib and

C5a peptidase are conserved proteins among different strains, which gives them an

advantage as new vaccine candidates [32].

Several researches have also focus on new therapies against S. aureus and S.

pneumoniae infections. As mentioned above, alpha-hemolysin, protein A and CPS have

been suggested as good targets for S. aureus infections [56]. For S. pneumoniae


infections, endopeptidase O (PepO) was suggested as a new vaccine candidate [124],

but also HtrA as a new possible target for vaccination [125].

However, although the search for new therapeutic targets is relevant and urgent,

the highest difficulty is to find a therapy that is effective against the different serotypes

and strains of the same bacterium. Based on this, the appropriate target appears to be

a structure that is highly conserved among different strains and serotypes of the same

pathogen, and ideally a structure conserved among different pathogens responsible for

the development of neonatal bacterial sepsis.

3.1 GAPDH

GAPDH is a well know glycolytic enzyme, essential for bacterial grown, but also

with virulent functions. Structurally, it is a tetramer composed by four identical subunits,

each one with approximately 45 kDa (Figure 5) [11].

Figure 5: GBS GAPDH. Structure GBS GAPDH NEM316, with the different subunits to be represented through four

colours. Structure PDB: 4QX6

Our group had identified GBS GAPDH as a VIP [11]. It is release upon cell lysis

and induces in the host a very fast production of IL-10. This result in an inability of

neutrophils to recruit into the site of infection [13]. It was also demonstrated, in mice, that

maternal vaccination with bacterial rGAPDH leads to a neutralization of IL-10 production

soon after infection, which results in an increase of neutrophil recruitment and protection

of neonates against GBS infections [13].

Moreover, early studies of our group demonstrated that neonatal mice deficient

in TLR2 are more resistant to GBS-induced sepsis than wild-type (WT) controls [12].

This virulence mechanism is in contrast with the current understanding of the

pathology of sepsis, which is usually explained by an increase in proinflammatory

cytokines and an excessive inflammatory response, including the production of

secondary mediators (such as reactive oxygen species (ROS)) and acute phase proteins

[126]. IL-10 is important in the negative balance of pro-inflammatory effects [127, 128].


19

Based on this, a study demonstrated that IL-10-/- animals have an exacerbated

proinflammatory cytokine response, which lead to the development of an irreversible

shock [128].

Nevertheless, although in the case of bacterial infections caused by GBS, IL-10

has been identified as an important mediator for the development of sepsis, the treatment

with IL-10 receptor inhibitors is not possible due to the simultaneously inhibition of other

important IL-10-dependent mechanisms. So, based on this and in the role of extracellular

bacterial GAPDH (bGAPDH) in the virulence mechanism of GBS infection and the fact

that its structure is highly conserved between different organisms, we hypothesized that

it might be a good target antigen for the prevention of neonatal sepsis. The potential of

GAPDH as a vaccine candidate is also recognized in other parasitical and bacterial

infections [129], including in S. pneumoniae infections [130].

GAPDH is a highly conserved protein between species, with protein alignment

studies demonstrating a homology of 40% between staphylococci, bovine and human

GAPDH. Thus, a vaccine based on this structure must take into consideration the host

GAPDH in order to avoid cross-immune reactions [129].

So, to avoid any possible cross-reactivity between human and bGAPDH, we

developed a vaccine constituted only by surface-exposed peptides of bGAPDH - PNV1.

Although, the peptides present in PNV1 aim to induce protective antibodies against six

different bacteria, the aim of this study is to evaluate the efficacy of PNV1 against three

different Gram-positive bacteria: GBS, S. aureus and S. pneumoniae.

3.1.1 B1 cells are responsible for IL-10 production upon bGAPDH stimulus

Previous study of our group had demonstrated that in GBS infections, GAPDH is

an important VIP, which induce a marked activation of B cells. Moreover, our group also

demonstrated that upon bacterial infection, an increase of the serum levels of IL-10 (an

immunosuppressive cytokine) occurs soon upon bacterial infection [11]. Previous work

included in the master thesis of Pedro Melo shows that when spleen mononuclear cells,

blood neutrophils, liver-derived macrophages and dendritic cells or splenic B cells from

newborn mice were stimulated with bacterial rGAPDH, B cells were the major source of

IL-10 upon rGAPDH stimulus when compared to the other neonatal leukocyte

populations in study (Pedro Melo, MSc in Biochemistry, 2013 and Figure 6).


Figure 6: Differences in IL-10 production between different population of cells. Spleen mononuclear cells (MNC),

neutrophils from peripheral blood (PMNC), liver macrophages (FLM) and dendritic cells (FLDC) and purified splenic B

cells from neonatal mice were stimulated in vitro with medium alone (RPMI), 0,5 µg/mL of LPS or 25 µg/mL of GBS

rGAPDH for 12h at 37⁰C, with 5% CO2. In all conditions were used 5x105 cells/well. Shown are the mean ± SD of three

independent experiments. Adapted from Pedro Melo, MSc Thesis in Biochemistry, 2013.

Moreover, when B cells from the spleen of neonatal mice were separated based

on surface expression of CD5, it was observed that CD5+ B cells (B1 cells) were the sub-

population that retained the ability to produce IL-10 upon bGAPDH stimulus (Pedro Melo,

MSc in Biochemistry, 2013 and Figure 7). This result suggests that B1 cells are the main

source of GAPDH-induced IL-10 production upon bacterial infection.


21

Figure 7: IL-10 production mediated by B cell populations. Spleen mononuclear cells (MNC), total B cells, B1 cells

and B2 cells from neonatal mice were stimulated in vitro with medium alone (RPMI), 0,5 µg/mL of LPS or 2,5 µg/mL of

GBS rGAPDH for 12h, at 37⁰C with 5% CO2. After that IL-10 was quantified in the supernatant of the cells. In all conditions

were used 2,5x105 cells/well. Shown are the mean ± SD of three independent experiments. Adapted from Pedro Melo,

MSc Thesis in Biochemistry, 2013.



23

Aim of the study

The present study was developed as a continuation of previous works of our

group which had demonstrated that GAPDH was an important VIP, which increases the

neonatal susceptibility to bacterial infections. Although the main focus of our group was

the neonatal infections caused by GBS, actually, we know that GAPDH is a conserved

structure between different bacteria. So, with this knowledge, we define the following

objectives:

- Evaluate the efficacy of a peptide-based vaccine that addresses bacterial

GAPDH (PNV1) in the prevention of neonatal sepsis caused by three Gram-

positive pathogens – GBS, S. aureus and S. pneumoniae.

- Characterize the cellular and molecular mechanisms associated with neonatal

susceptibility to bacterial infections caused by the referred bacteria.



25

Material and Methods

Chapter 2



27

Bacterial Strains and Growth conditions

In the present study three Gram-positive bacterial strains were used: GBS, MRSA

and S. pneumoniae. All the strains correspond to clinical isolates obtained at Centro

Hospitalar do Porto – Table 1. The Microbiology Department provided all the bacteria

strains. GBS and MRSA were grown in TH broth or agar (Difco Laboratories), containing

10 mg/L of colistin sulphate and 5 mg/L of oxalinic acid (Streptococcus Selective

Supplement, Oxoid). S. pneumoniae was grown in TH broth and yeast extract (2% (m/v)),

supplemented with 10 mg/L of colistin sulphate, 5 mg/L of oxalinic acid and 10 mg/L of

choline chloride or blood agar medium (Oxoid). Bacteria were grown at 37⁰C.

Table 1: List of bacteria and respective strains used during the experiments.

Bacteria Strain

Streptococcus agalactiae, GBS Serotype III Erythromycin resistant

Staphylococcus aureus MRSA

Streptococcus pneumoniae Penicillin-resistant

Animals and Ethics statements

Six-to eight-week old male and female C57BL/6 and TLR2-deficient

C57BL/B6.129-Tlr2tm1Kir/J (TLR2-/-) mice were purchase from the Jackson Laboratory and

were quarantined for a week prior to study infection. The animals were kept at the

Instituto de Investigação e Inovação em Saúde (i3S) Animal Facility during the

experiments. Animals were housed in techniplast ventilated polycarbonate cages under

positive pressure with hard-wook bedding and controlled environment (40 air changes

per hour, temperature between 21-23 ⁰C, relative humidity at 55±10% and 12 h light/dark

cycle). The Mucedola Diet and water was provided ad libitum. Mice were identified by

ear tag.

All procedures were performed according to the European Convention for the

protection of Vertebrate Animals used for Experiment and Other Scientific Purpose (ETS

123) and 2010/63/EEC Directive and Portuguese rules (DL 113/2013). All animal

experiments were planned to minimize mice suffering, and during experiments,

newborns mice were kept with their mother.


Preparation of Neonatal Vaccine

Neonatal vaccine (PNV1) is composed of three surface-exposed peptides from

bGAPDH that are not present in human GAPDH. The choice of the peptides was based

on Multiple Alignment and Sequence Similarity percentages from the ClustalW2 server,

after submitting bGAPDH amino-acid sequences (FASTA format). Peptides were

synthesized at Biomatik (acetate salt, 95% purity in HPLC) and the amino-acid

sequences of each peptide are as follows:

Peptide 1 (Sequence from N to C): RIQEVEGLEVTR 12aa

Peptide 2 (Sequence from N to C): DVTVEEVNAAM 11aa

Peptide 3 (Sequence from N to C): EVKDGHLIVNGKK 13aa

C57BL/6 mice were immunized intraperitoneally (i.p.) four times with PNV1 (with

a two-week interval between doses). Three different doses were tested, with formulation

containing equal amounts of the different peptides (20 µg, 2 µg or 0.2 µg) in a 1:8 PBS-

Alhydrogel suspension in a total volume of 200 µL. Sham-immunized control animals

received 200 µL of 1:8 PBS-Alhydrogel suspension (Vehicle control with Alhydrogel).

Rats and rabbits were immunized with 0.2 µg of each peptide. Antibodies titers were

determined by ELISA assay as described below.

Neonatal mouse model of infection

Infection studies were performed in C57BL/6 (WT) or TLR2 knockout (TLR2-/-)

mice, with less than 48 hours of life. Mice were infected subcutaneously (s.c.) with

6.6x105 CFU of GBS, 3.6x105 CFU of MRSA or 2.0x106 CFU of S. pneumoniae, in a

maximum volume of 40 µL. During the entire length of the experiment the animals were

kept with their mother and monitored daily.

Purification of anti-PNV1 IgGs

Rats and mice were immunized i.p. four times with 0.2 µg of PNV1 (with a two-

week interval between doses). PNV1 IgGs were purified from pooled sera of immunized

animals using a HiTrap Protein G affinity column (HiTrap, GE Healthcare BioSciences

AB). Purified IgG antibody fractions were further equilibrated in PBS, aliquoted and

stored at -80ºC.

PNV1 IgG passive immunizations

Antibody treatment were performed in newborn C57BL/6 (<48h), 12h prior to

bacterial infection. For passive immunization newborn mice were injected s.c. with 150


29

µg of anti-PNV1 IgG antibodies, in a maximum volume of 100 µL. Control animals

received the same amount of control IgG.

Quantification of serum IgG

The antibody production was evaluated by an ELISA assay. Briefly, polystyrene

microtiter plates (Nunc MaxiSorp 96 well plates) were coated overnight at 4⁰C with 5

µg/mL of indicated protein, in a final volume of 50 µL per well. The plates were then

saturated for 1h with 1% Bovine Serum Albumin (BSA) (VWR) in PBS at room

temperature. Appropriate serum dilutions (performed in 1% BSA in PBS) were incubated

in the plates for 2h at room temperature. After that, the plates were washed five times

with PBS, and incubated for 1h with 50 µL of antibody – Goat Anti-Mouse IgG, Human

ads-BIOT (Southern Biotech), with further incubation of detection antibody –

Streptavidin-horseradish peroxidase (HRP) (Southern Biotech). After washing, 100 µL

of 3,3`,5,5`-tetramethylbenzidine (TMB) (Southern Biotech) was added and used as

substrate. 1 M of sulfuric acid (H2SO4) (Merck Millipore) was added to stop the reaction,

and the absorbance was read at 450 nm. IgG titers were defined as the first value of

sample dilution where the OD450≤ 0.1.

Purification of the bacteria rGAPDH

For production of rGAPDH protein from GBS, E. coli, S. aureus, S. pneumoniae

and Klebsiella pneumoniae (K. pneumoniae), E. coli BL21 (DE3) strains (Novagen) and

the pET28a plasmid (Novagen) were used, as described previously [11]. E. coli was

cultured on Luria-Bertani (LB) (Sigma Aldrich)) medium, containing 0.5 mM Isopropyl β-

D-1-thiogalactopyranoside (IPTG) (Sigma-Aldrich), 16 h at 20 ⁰C. After that, the pET28

derivative cells were harvested by centrifugation for 30 minutes at the maximum velocity

and processed in order to disrupt the cell integrity (Emulsiflex). The soluble fraction was

isolated by ultracentrifugation and applied to a His-Trap HP column (Amersham

Biosciences). An imidazole (Sigma Aldrich) gradient (50 mM, 300 mM and 500 mM) was

used and rGAPDH was eluated with 300 mM imidazole. A second purification was

performed by size-exclusion chromatography (S200 26/60 GE Healthcare) using PBS as

mobile phase. A Native-Page analysis was performed in order to confirm rGAPDH

purification. The thermal stability was evaluated by Differential Scanning Calorimetry and

enzymatic activity was studied using a specific kit – GAPDH Activity Assay (Abcam).


Crude extracellular proteins (CEP)

Bacterial strains were grown in LB medium because TH medium naturally

contains a high amount of proteins. A pre-grown was performed overnight at 37⁰C and

after that, bacteria were grown during 6h at 37⁰C. The medium was centrifuged 20 min

at 4⁰ C, 3500 g. The supernatant was filtered through a 1.2 µm, 0.45 µm and 0.2 µm pore

size filter (Merck Millipore). The sample was concentrated by dialysis and reserved at 4⁰

C.

Western Blot analysis

The presence of GAPDH in the CEP of GBS, MRSA and S. pneumoniae, and the

recognition of bacterial rGAPDH and human rGAPDH was performed by Western Blot

(WB) analysis, using PNV1-elicited antibodies and control mice serum.

For that purpose, GBS rGAPDH, S. aureus rGAPDH, S. pneumoniae rGAPDH,

E. coli rGAPDH, K. pneumoniae rGAPDH, human rGAPDH and CEPs of GBS, MRSA

and S. pneumoniae were applied to a 10% SDS-PAGE. The proteins were transferred

onto a PVDF membrane (Immobilon-P Millipore) and stained with Ponceau S. After de-

staining with water, the membrane was saturated for 1 hour with TBST buffer (0,01 M

Tris (Merck Millipore), 0,15 M NaCl (VWR), 0,05% Tween 20 (VWR (pH 8.0)), containing

1 or 3% of BSA, and further incubation overnight at 4 ⁰C (with mice anti-PNV1 IgG and

control mice serum in TBST+3%BSA), or during 2 hours (with rat anti-PNV1 IgG in

TBST+1%BSA) diluted 1:500. After that, it was incubated for 1 hour with appropriate

secondary antibody diluted 1:10000 in TBST+3% BSA (HRP-coupled monoclonal goat

anti-mouse IgG (Southern Biotech)) or 1:1000 in TBST+1% BSA (Mouse Anti-Rat IgG1-

UNLB (Southern Biotech) and further incubation with Goat Anti-Mouse Ig (H+L)-AP

(SouthernBiotech)). The detection was done using a solution of ECL, a luminol-based

chemiluminescent substrate for the detection of HRP, with the chemiluminescent

detection performed using the Chemidoc (Bio-Rad), or with nitro-blue tetrazolium

chloride (NBT) and 5-bromo-4-chloro-3`-indolyphosphate p-toluidine salt (BCIP) in

alkaline phosphate (AP) buffer (0,01M Tris, 0,01M NaCl, 0,5mM MgCl2 (Merck Millipore)

(pH 9.5)), as substrate.

Cell cultures

1. B1 cell purification

The spleen of newborn mice (<72 h) were aseptically collected and homogenized

in complete RPMI (cRPMI) – RPMI 1640 supplemented with penicillin (100 IU/mL),


31

streptomycin (50 µg/mL), 2-mercaptoethanol (2-ME) (0.05 M) and 10% Fetal Bovine

Serum (FBS) (Sigma-Aldrich). B1 cells were purified by magnetic cell sorting using a

Mouse B1 cell Purification Kit (Miltenyi Biotech) according to manufacturer’s instructions.

This strategy allows for the isolation of this cell population with 95% purity. Cells were

then distributed in 96-well plates (2.5x105 cells/well) and exposed to five different

conditions: medium alone, medium containing 25 µg/mL of GBS rGAPDH, medium

containing 10 µg/mL of TLR inhibitors - OxPAPC (TLR2 and TLR4 signalling inhibitor)

and CLI095 (TLR4 signalling inhibitor) and medium containing 1 µg/mL Pam3CSK4

(Invivogen). Cells were cultured for 12h at 37 ⁰C in a humidified atmosphere containing

5% CO2.

2. IL-10 quantification

IL-10 quantification from the supernatant of mouse cell culture was performed by

ELISA according to the manufacturer’s instructions (R&D Systems).

Statistical analysis

All statistical analysis was performed in GraphPad Prism version 5.0 software.

Survival trials were analysed with log-rank test.



33

Results

Chapter 3



35

PNV1 immunogenicity

In order to evaluate the immunogenicity of our peptide-based vaccine (PNV1),

composed of surface-exposed peptides present in the GAPDH from GBS, S. aureus and

S. pneumoniae, C57BL/6 mice were immunized four times with three different doses of

PNV1 (with a two-week interval between doses). As shown in the Figure 8, the dose

containing 0.2 µg of each peptide were the most immunogenic, inducing high antibody

titers specific for Gram-positive (GBS) and Gram-negative (E. coli) bacterial rGAPDH.

Figure 8: Serum anti-GAPDH IgG antibodies in mice immunized with PNV1. C57BL/6 mice were immunized i.p. four

times (with a two-week interval between doses) with 200 µL of three different doses of PNV1 (20.0 µg, 2.0 µg and 0.2 µg)

formulated in a 1:8 PBS-Alhydrogel suspension. Control animals received the same amount of 1:8 PBS-Alhydrogel

suspension. Antibody titers were determined by ELISA assay. Shown is the mean ± SEM of three independent

experiments. For each group, at least 9 animals were used.

Based on the results presented above, we decided to immunize rabbits and rats

with the dose that showed to be most immunogenic in mouse, using the same

immunization schedule.

As shown in the Figure 9, the formulation containing 0.2 µg of each peptide was

also immunogenic in these animals, as demonstrated by the elevated levels of antibody

production after immunization, when compared to the control group.

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

Control A20.0 A2.0 A0.2

IgG

tit

ers

anti-GAPDH IgG

Gram (+) Gram (-)


Figure 9: Serum anti-PNV1 IgG antibodies titers in rats and rabbits immunized with PNV1. Rats and rabbits were

immunized i.p. four times (with a two-week interval between doses) with equal amounts of the different peptides (0.2 µg)

that compose PNV1, formulated in 1:8 PBS/Alhydrogel suspension. Control animals received the same amount of 1:8

PBS-Alhydrogel suspension. Antibodies titters were determined by ELISA assay. Shown is the mean ± SEM of four

independent experiments, with a minimum of 5 animals per group.

PNV1-elicited antibodies react with extracellular bacterial

GAPDH

GAPDH has a higher homology between different bacterial pathogens and based

on this, PNV1 appears as an efficient alternative in the prevention and as possible

0,0

0,5

1,0

1,5

2,0

2,5

3,0

0 5 10

Abs 4

50nm

log dil

Rat

Control

PNV1

0,0

0,5

1,0

1,5

2,0

2,5

0 5 10

Ab

s 4

50

nm

log dil

Rabbit

Control

PNV1


37

therapy against bacterial infections responsible for the development of sepsis. As

mentioned before, to avoid any cross-reactivity between human and bGAPDH, PNV1 is

constituted only by surface peptides of bGAPDH. Based on this, next we tested by WB

the recognition of rGAPDH from five different bacteria by PNV1-elicited antibodies. As

observed in Figure 10B PNV1 IgG antibodies were able to recognize rGAPDH from GBS,

S. aureus, S. pneumoniae, K. pneumoniae and E. coli. However, human rGAPDH (used

as a control) was also recognized by PNV1 IgG. Nevertheless, when we used purified

IgG from the sera of control mice, we also observed a band in the WB corresponding to

the recognition of human rGAPDH (Figure 10A), indicating that antibodies against human

GAPDH were not elicited by PNV1 vaccination. To confirm this, we perform an ELISA to

compare the antibody titers against human rGAPDH in PNV1-immunized versus control

animals. As observed in Figure 11, PNV1 immunization did not induce an increase in the

titers of IgG specific for human GAPDH.

Additionally, we performed a WB analysis to evaluate if PNV1 IgG were able to

recognize extracellular GAPDH from the CEP of GBS, MRSA and S. pneumoniae. As

shown in Figure 12, PNV1 IgGs were able to recognize GAPDH in the CEP of GBS,

MRSA and S. pneumoniae.

Figure 10: Western Blot analysis of Gram-positive, Gram-negative and human rGAPDH. Western Blot analysis of

blotted protein samples corresponding to GBS rGAPDH, MRSA rGAPDH, S. pneumoniae (Pneumo) rGAPDH, E. coli

rGAPDH, Klebsiella pneumoniae (Kleb) rGAPDH and human rGAPDH. In all cases were used 1 µg of recombinant protein.

(A) Control mice serum in a 500-fold dilution were used as developing Abs. (B) PNV1-elicied antibodies purified from mice

serum immunized with PNV1 were used in a 500-fold dilution as developing Abs.

A B


Figure 11: Serum anti-Human GAPDH IgG in mice immunized with PNV1. C57BL/6 mice were immunized i.p. four

times (with a two-week interval between doses) with 0.2 µg of PNV1 formulated in a 1:8 PBS-Alhydrogel suspension.

Control animals received the same amount of 1:8 PBS-Alhydrogel suspension. Antibodies titers were determined by

ELISA assay. Shown is the mean ± SEM of two independent experiments.

Figure 12: PNV1-elicited antibodies were able to recognize bacterial GAPDH in the bacterial CEP. Western blot

analysis of blotted protein samples corresponding to GBS rGAPDH (positive control), CEP-GBS, CEP-MRSA and CEP-

S. pneumoniae (CEP-Pneumo). PNV1-elicied antibodies purified from rat serum immunized with PNV1 were used in a

500-fold dilution as developing Abs.

Altogether, these results indicate that animals immunized with PNV1 are able to

produce specific antibodies that recognize extracellular GAPDH of GBS, S. aureus and

S. pneumoniae.

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

Control PNV1 imnz

IgG

tit

ers

anti- human GAPDH IgG


39

TLR2 signalization is required for IL-10 production mediated by

B1 cells upon bacterial GAPDH

TLR2-induced IL-10 production is a risk factor for neonatal bacterial sepsis. This

was first demonstrated by the fact that WT animals were more susceptible to GBS

infections than TLR2-/- mice and that TLR2-/- pups have low levels of serum IL-10 soon

after infection [12].

Thus, based on the high levels of IL-10 production observed in B1 cells in

response to rGAPDH stimulus, and the fact that TLR2-/- mice have decreased IL-10

production and increased survival than WT animals upon GBS infection, we hypothesize

that TLR2 is the cellular receptor for bGAPDH. In order to confirm our hypothesis, B1

cells from the spleen of newborn mice were stimulated with rGAPDH in the presence of

TLR2 or TLR4 inhibitors. As shown in the Figure 13A, the production of IL-10 GAPDH-

induced by B1 cells, was blocked if a chemical inhibitor of the TLR4 and TLR2 signalling

pathway (OxPAPC) was added to the cultures. Nevertheless, when an inhibitor of only

the TLR4 signalling pathway was added (CLI095), B1 cells retained the ability to produce

IL-10 upon GAPDH stimulus (Figure 13A). This indicates that in the absence of TLR2

signalling, B1 cells are not able to produce IL-10 in response to bGAPDH.

To confirm this, B1 cells were isolated from the spleen of WT and TLR2-/- neonatal

mice and both were stimulated in vitro with bGAPDH. As depicted in Figure 13B, B1 cells

from TLR2-/- pups lost their ability to produce IL-10 in response to bGAPDH, in clear

contrast to what is observed in B1 cells from WT mice. Again, this result indicates that

TLR2 is the cellular receptor for bGAPDH, responsible for the early IL-10 production

observed in neonatal GBS infections.

OxPAPC

A


Figure 13: GAPDH-mediated IL-10 production in mice B1 lymphocytes is blocked when TLR2 signaling is

inhibited. (A) B1 cells purified from the spleen of newborns mice were stimulated in vitro with rGAPDH in the presence

of Cli095 (TLR4 signalling inhibitor) or OxPAPC (TLR2 and TLR4 signalling inhibitor) for 12h at 37⁰C, with 5% CO2. 2.5x105

cells/well were used. IL-10 was quantified after the incubation period. (B) WT or TLR2-/- B1 cells purified from the spleen

of WT and TLR2-/- mice, respectively, were stimulated in vitro with Pam3CSK4 (TLR1/TLR2 agonist) or rGAPDH for 12h

at 37⁰C, with 5% CO2. 2.5x105 cells/well were used. IL-10 was quantified after the incubation period. Shown are the mean

± SD of at least four independent experiments.

TLR2 KO animals have an increased survival upon bacterial

infection

Based on the results obtained in vitro, we decided to initiate in vivo studies.

Previous studies by our group had demonstrated that TLR2-/- animals showed increase

survival when compared to WT animals, after infection with GBS NEM316 [12]. Thus,

our aim was to evaluate if the same type of protection occurs in infections caused by

MRSA and S. pneumoniae. For this, the neonatal mice were infected and the survival

curves were determined in a 12-day period following infection.

As observed previously with GBS NEM316 infections [12], TLR2-/- mice are more

resistant to MRSA and to S. pneumoniae infections than the WT mice (Figure 14).

WT B1 cells

TLR2-/-

B1 cells

B


41

Figure 14: TLR2-/- newborns mice have an increased of survival upon bacterial infection. TLR2-/- and C57BL/6 (WT)

newborns mice (<48h) were infected with MRSA or S. pneumoniae (A) TLR2-/- and WT mice were infected with 3.6x105

CFU of MRSA (B) TLR2-/- and WT mice were infected with 2.0x106 CFU of S. pneumoniae. In both cases were used a

maximum volume of 40 µL and survival curves were determined in a 12-day after infection. During the experiment time,

newborns mice were maintained with their mother. In all the results, the numbers between parentheses represent the

number of animals that survived to bacterial infection versus the total number of infected animals. Statistical differences

(P value) between TLR2-/- and control animals are indicated. The results represent data pooled from three independent

experiments.

A

B


PNV1 as an efficient therapy against bacterial infection

Previous results from our group had demonstrated that GAPDH is an important

virulence factor in bacterial infections caused by GBS [11, 13]. Although not yet fully

described the cellular and molecular mechanisms associated with GAPDH-induced

susceptibility to GBS infections, we hypothesize that it could be a conserved mechanism

of immune evasion among different bacterial pathogens. In fact, GAPDH is highly

conserved between both Gram-positive and Gram-negative bacteria associated with

neonatal sepsis.

Thus, we decided to test the efficiency of PNV1-elicited IgGs in neonatal

infections caused by GBS, MRSA and S. pneumoniae. For that purpose, pups were

passively immunized with PNV1 IgGs, 12h before the challenging bacterial infection.

Survival was compared with infected pups that received control IgGs. Interestingly, the

group of animals that received PNV1 IgGs presented a significant increased survival

when compared to controls (Figure 15).

GBS Serotype III A


43

Figure 15: PNV1 immunization protects newborn mice against bacterial infection. C57BL/6 mice (<48h) were

immunized with 150 µg of PNV1-elicited antibodies (PNV1-IgG) or with control-elicited antibodies (Control IgG) in a

maximum volume of 100 µL, and infected with GBS, MRSA or S. pneumoniae. (A) Newborns mice were infected with

6.6x105 CFU of GBS. (B) Newborns mice were infected with 3.6x105 CFU of MRSA. (C) Newborns mice were infected

with 2.0x106 CFU of S. pneumoniae. In all the cases mice were infected with a maximum volume of 40 µL and survival

curves were determined in a 12-day after infection. During the experiment time, newborns mice were maintained with their

mother. In all the results, the numbers between parentheses represent the number of animals that survived to bacterial

infection versus the total number of infected animals. Statistical differences (P value) between the two groups are

indicated. The results represent data pooled from at least two independent experiments.

Altogether, these results indicate that PNV1 can be an efficient vaccine against

neonatal infections caused by GBS, S. aureus or S. pneumoniae.

B

C



45

Discussion

Chapter 4



47

Severe sepsis and septic shock caused by bacterial infections are a major

healthcare problem [131]. Newborns are particularly susceptible to such infections, with

WHO estimating that, per year, more than 1 million deaths occur due to neonatal sepsis

[1].

At birth, newborns suffer a dramatic transition from mother’s womb to a non-

sterile outside environment and are exposed to a variety of potentially pathogenic

microorganisms that were not found in utero. At this stage, adaptive immune system is

not fully developed, so newborns must rely on their innate immune system to control

infections that can possible occur. The sterile intrauterine environment ensures that

newborns are not exposed to pathogenic microorganisms, so at birth there is no

immunological memory against specific antigens. Based on this, maternal antibodies

passively transferred across the placenta have a crucial importance, although it

represents a finite protection against pathogens [132].

Neutrophils are the first cells to be recruited to the site of infection. However,

studies had demonstrated that in neonates their recruitment is impaired due to a

decrease in the L-selectin expression and to a lower capacity to upregulate CR3 (also

known as CD11b/CD18 or Mac-1) on the neutrophil surface [115, 116].

Neonatal sepsis can be caused by a variety of pathogens, including viral,

bacterial and fungal microorganisms [14, 81]. However, bacterial sepsis has a particular

importance, being the seventh-leading cause of neonatal death in the United States

[133].

Neonatal infections can be transmitted before, during or after birth. GBS,

Staphylococcus spp. and E. coli contribute to 58% of the cases of neonatal sepsis.

However other pathogens should be considered, such as, K. pneumoniae,

Pseudomonas aeruginosa and S. pneumoniae [4, 5].

In the present work we focused our study in GBS, MRSA and S. pneumoniae

(Gram-positive pathogens). These are three well described microorganisms in the

context of neonatal bacterial sepsis. S. pneumoniae has a lower incidence, but has a

high mortality rate associated [60]. Previous studies of our group had focused in neonatal

sepsis caused by GBS, describing GAPDH as an important virulent factor. As a

continuation of this work, we decided to extend our study to other important bacteria

responsible for the development of sepsis.

Considering that the cellular and molecular mechanisms associated with

neonatal susceptibility to bacterial infections is not fully characterized, we define this as

one of our objectives. Another important aim of our work was to evaluate the

effectiveness of PNV1 as a vaccine for neonatal bacterial sepsis.


TLRs are important pattern-recognition receptors, which are able to recognize

bacterial structures called PAMPs [118]. Recognition of PAMPs leads to the production

of signals which result in cell activation and contribute for an effective host defence.

Nevertheless, some microbes can engage different pattern recognition receptors in order

to subvert host immunity [118]. Previous published data demonstrates that during

Yesteria enterocolitica and Candida albicans infections occurs an increase of IL-10

production mediated by TLR2, which increase the host susceptibility to these infections

[134-136]. Based on this, we decided to start our study by the research of the cell

population(s) responsible for the production of IL-10 upon bGAPDH stimulus and for the

evaluation of TLR2 as a possible receptor for bGAPDH. As demonstrated in the results

presented in the Figure 7, CD5+ B1 cells are the main responsible for IL-10 production

upon bGAPDH stimulus, while B2 cells (CD5-) produce only traceable amounts of this

immunosuppressive cytokine. We also demonstrated here that TRL2 can be the

bGAPDH receptor on the surface of B1 cells, based on the observation that B1 cells

exposed to chemical inhibitors of TLR2 and/or TLR4 signalling pathway lost their ability

to produce IL-10 upon in vitro rGAPDH stimulus (Figure 13A). This same result was

confirmed by using B1 cells from TLR2-/- mice pups and by comparing their ability to

produce IL-10 upon bGAPDH stimulus with B1 cells from WT mice (Figure 13B).

Immunotherapies in this area have become crucial, because actually there is no

available effective measure for the prevention or treatment of neonatal sepsis. The

current treatment is based on antibiotic administration, however the emergence of multi-

resistant bacterial strains is continuously being described [122]. IAP is the prophylactic

measure used in the prevention of GBS infections, which since their implementation,

lead to a decrease in the incidence of EOD, but not in the frequency of LOD. In addition,

this measure increased the number of infections caused by Gram-negative pathogens,

in particular infections caused by E. coli [81]. 13-valent PCP (Prevnar 13) is used for the

prevention of S. pneumoniae infections in infants, however, there is still no vaccine

licensed to address pneumococcal infections in the newborn. In addition, Prevnar 13

formulation is based on the most common serotypes in North America and includes only

13 of the 93 serotypes identified, which does not confer protection against all S.

pneumoniae strains known to be pathogenic in humans. So, based on the increase

susceptibility of newborns to infections, due to their undeveloped immune system, the

maternal vaccination appears to be the most promising alternative [121].

GAPDH is a protein present in many microorganisms, essential in metabolic

pathways, specifically in glycolysis and for bacterial growth, but also with virulent

functions, with previous reports recognizing GAPDH as a “moonlighting protein” due to

it’s localization at the bacteria surface or as an excreted protein [11]. This surface


49

localization for a bGAPDH was first reported for S. pyogenes GAPDH. Posteriorly,

GAPDH has been described as a surface or excreted protein in different bacteria such

as in Streptococci from groups A, B, C, E, G, H and L [137, 138] and in Gram-negative

pathogens such as enterohemorrhagic and enteropathogenic E. coli [139]. Other

“moonlighting proteins” have been identified, such as glucose-6-phosphate isomerase,

ornithine carbamoyltransferase, phosphoglycerate kinase [140], α-enolase [141], and

fibronection-binding proteins (such as PavA of S. pneumoniae) [142]. The protective

effect of this proteins as target antigens to the development of new vaccines have also

been considered as the example of fructose-biphosphate aldolase of S. pneumoniae

[130], α-enolase of Streptococcus sobrinus and Streptococcus suis [143, 144].

After identifying GAPDH as an extracellular protein in S. aureus and S.

pneumoniae, which in the later was also described by others [145], we aimed to evaluate

if extracellular GAPDH was also an active VIP in these bacteria. To confirm this, we took

advantage of knowing the cellular receptor for GAPDH – TLR2. Interestingly, we were

able to find that TLR2-/- neonatal mice were much more resistant against MRSA and S.

pneumoniae infections than WT controls (Figure 14). Previous studies also described an

increased resistance of TLR2-/- mice against infections caused by Candida albicans [134]

and GBS infections [12]. In all of these cases, the lack of TLR2 signalling was also

associated with decreased IL-10 production and thus with protection.

Based on this, we decided to evaluate the possible use of PNV1 as a preventive

and therapeutic measure for neonatal bacterial infections. We began our work for the

evaluation of PNV1 immunogenicity. For that, three doses were tested (0.2 µg, 2 µg and

20 µg of each peptide) in mice. From the results obtained, we found that the dose of 0.2

µg was the most immunogenic in mice (Figure 8). The immunogenicity of PNV1 was also

observed in rats and rabbits (Figure 9). After that and taking in consideration that GAPDH

is an extracellular virulent factor in GBS infections, we assess if this could happen in

other Gram-positive bacteria. The presence of an extracellular protein recognized by

PNV1 IgGs was found in the CEP of three clinical isolates, namely GBS, MRSA and S.

pneumoniae (Figure 12). Although this result strongly indicates the presence of GAPDH

as an extracellular protein also in S. aureus and S. pneumoniae, this should be confirmed

by protein sequencing. For the case of MRSA, the presence of a band at a higher and

lower molecular weight was also verified. In our point of view the first one may

correspond to a protein dimer, while the lower molecular weight band may correspond

to protein degradation.

PNV1 vaccine was developed based on the peptide sequence displayed in

different bGAPDH, whereby the aim was to evaluate its use not only in one type of

bacterial infection, but in neonatal infections caused by various pathogens. Thus by WB


analysis we decided to evaluate the recognition of different bGAPDH as well as the

human GAPDH, using PNV1-elicied antibodies and control mice serum. As can be seen

from the results shown in Figure 10B, PNV1-elicited antibodies were able to recognized

GBS, S. aureus, S. pneumoniae, E. coli and K. pneumoniae rGAPDH, as well as human

rGAPDH. However, this recognition of human rGAPDH was also observed when control

mice serum was used. The same was not true with the recognition of bacterial rGAPDH

(Figure 10A). However, in order to clarify this situation, we decided to perform an ELISA

assay to compare the IgG antibody titers against human GAPDH in PNV1-immunized or

control mice. As shown in the Figure 11, PNV1 immunization did not induce an increase

in the titers of IgG specific for human IgG. The presence of antibodies in the sera of

control mice able to recognize human GAPDH may be due to a cross-reactivity of the

“physiological” auto-antibodies against mice GAPDH present in the sera of these

animals. Thus, the results suggest that the peptides present in PNV1 formulation have

homology with GBS, S. aureus, S. pneumoniae, E. coli and K. pneumoniae rGAPDH but

not with human GAPDH, being safe for use in humans. To confirm the protective effect

of PNV1 against neonatal infections, we decided to proceed with the first in vivo trials.

To do this, we performed passive immunization of newborn mice (<48h) with PNV1-IgGs

or control IgGs. As can be seen in the Figure 15 newborns passively immunized with

PNV1 IgGs showed an increased survival upon GBS, MRSA and S. pneumoniae

infection, when compared to the control group.

The results presented herein are important since, as mentioned above, currently

there is no type of preventive measures effective for neonatal infections against different

microorganisms. As can be seen in the literature, many groups have been dedicated to

the search of new virulence factors that can be used as therapeutic targets for the

development of vaccines. For S. pneumoniae new virulence factors have been found,

such as serine protease HtrA that help bacteria to survive in the presence of

environmental pressures like elevated temperatures [125], but also in the cell division

[146]. Studies showed that in models of pneumoniae and bacteremia, HtrA deficient

pneumococci had a lower virulence [125]. Another virulent factor identified and

suggested as a possible adjuvant vaccine was PepO, an agonist of TLR2 and TLR4.

Studies demonstrated that knockout animals for TLR2 and TLR4 treated with

recombinant PepO (rPepO) had a lower production of cytokines, lower neutrophil

recruitment and increased tissue damage when compared with the WT mice [124]. In

relation to S. aureus infections, many new therapeutic targets have been identified, and

currently nine antibiotics are in clinical trials, of which one is in phase I and eight in phase

II [56]. The research for new clinical treatments is very important due to the presence of

S. aureus strains also resistant to vancomycin, the most used antibiotic against MRSA


51

and multi-drug-resistant S. aureus [53]. The use of quinolones has been advanced as a

strategy for the treatment of such infections, but also oxazolidione class and fatty acid

synthesis inhibitors. Nevertheless, and again, there is no preventive strategy available

against these infections.

In terms of target antigens, the literature also refers classical approaches such

as protein A (staphylococcal antibodies against surface proteins) and CPS (strategy also

suggested for GBS infections) [56]. Protein A has the ability to bind the immunoglobulin

Fcɣ domain, which inhibits the opsonophagocytic killing of S. aureus, and to bind the Fab

domain of V3H-type B cell response (IgM) [147]. Passive immunization with monoclonal

antibodies against protein A led to an increase survival of neonatal mice to S. aureus

infections [148]. Addressing S. aureus CPS appeared to be a possible therapeutic

strategy, as occurred with GBS infections, especially because in the case of S. aureus

infections it is known that 70 to 80% are caused by one of two serotypes, 5 (CPS5) or 8

(CPS8) [149]. Active immunization with capsular conjugated vaccine or passive

immunization with CPS-specific was able to reduce the severity of infection in animal

models [150, 151]. However, these effects were not found in humans, since a vaccine

based on anti-CPS types 5 and 8 failed in the phase II clinical trials [152, 153].

For GBS infections, the literature considers the use of CPS as a therapeutic

strategy, but also the use of antigens of the pili structure [46, 154]. However, in the first

case, and despite being already in clinical trials a vaccine, it is known that this does not

give protection against all serotypes [49]. In the second case, although the pili has a very

conserved structure between different GBS strains, studies in Neisseria gnorrhoeae

showed antigenic variations, which put in question the development of a vaccine based

on this structure [155]. Other proteins have been suggested as good vaccine targets,

such as Rib, Sip and C5a peptidase [32]. The results obtained in vivo with animal models

suggest that PNV1 can be considered as a possible therapeutic strategy not only in GBS

infections, but also in infections caused by others pathogens, since it was based on a

protein structure - GAPDH - which is very conserved among different serotypes of

different bacterial pathogens.

In conclusion, the present thesis suggests a new vaccine candidate against

bacterial infections caused by Gram-positive pathogens. Moreover, it helps to clarify the

cellular and molecular mechanism associated with the development of neonatal

susceptibility to such infections, identifying TLR2 as the possible receptor for bacterial

GAPDH on the surface of B1 cells, which leads to a rapid production of high IL-10 levels

upon bacterial infection. Thus, PNV1 vaccination appears to be a good strategy to

prevent neonatal bacterial infections (Figure 16).


Figure 16: PNV1-elicied antibodies protection against neonatal bacterial infections. bGAPDH is recognized by TLR2

in the surface of B1 cells, which leads to an increase of IL-10 production and consequent impair in neutrophil recruitment,

bacterial proliferation and septic shock development (red diagram). Passive immunization with anti-PNV1 IgGs appears

to prevent the binding of bGAPDH to TLR2 receptor on the surface of B1 cells, which confer protection due to the less IL-

10 production, that results in an increase of neutrophil recruitment and consequent bacterial clearance (green diagram).


53

Future works

Chapter 5



55

Based on the results obtained, future works should focus on the exact

understanding of the virulence mechanism of the different pathogens in study, which

results in the increase susceptibility of newborns to bacterial infections. This

comprehension is important because, although there are already data for GBS infection

supporting GAPDH as an important virulent factor associated with an increase of IL-10

production, it has not been found for the others pathogens under study. Thus, it becomes

essential to test if the mechanism of infection is conserved among different agents in

order to give further support to GAPDH as a major virulent factor, and PNV1 as a novel

global therapeutic strategy.

Also, considering the broad spectrum of agents responsible for the development

of neonatal sepsis, future studies should also focus on the evaluation of PNV1 as a

therapeutic strategy for neonatal bacterial infections caused by other agents, including

Gram-negative bacteria. Furthermore, knowing that there are other groups at risk for

developing bacterial sepsis, as the elderly and people with chronic diseases such as

diabetes, futures studies may also cover these groups.

Finally, it becomes equally important to determine if the mechanisms of virulence

observed in vitro in the present study using mice cells are conserved in human cells. This

study is essential, since the final objective of PNV1 is to be used as a therapeutic strategy

in humans.



57

Conclusion

Chapter 6



59

Neonatal infections are a major cause of death. The treatments available to

address this problem are based only on antibiotic administration.

Previous studies of our group had identified GAPDH as an important virulent

factor in GBS infections. However, considering that GAPDH is a highly conserved protein

also present in other relevant pathogens, we decided to focus our study not only in GBS,

but also on S. aureus and S. pneumoniae.

The results obtained suggested that TLR2 is the receptor on the surface of B1

cells that recognize bGAPDH, and that these cells are the major source of IL-10 upon

GAPDH recognition. Based on this, we performed infection studies in TLR2-/- mice using

MRSA and S. pneumoniae, which demonstrated increased resistant to bacterial

infections when compared to WT animals.

In addition, our work advances a new vaccine (PNV1) candidate as a preventive

strategy against neonatal bacterial infections, that can be caused by various pathogens,

since the formulation is based on peptides present in the surface of different bGAPDH,

which are not present in human GAPDH.

In order to confirm this, we performed different studies, which demonstrated that

prior immunization with PNV1-elicited IgGs conferred protection of newborns against

GBS, S. aureus and S. pneumoniae infections. However, future trials should be done

with other bacterial pathogens, including Gram-negative microorganisms, in order to

evaluate the possible use of PNV1 as a preventive strategy for a large spectrum of

agents responsible for such infections.



61

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63

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