Lassa Virus and the Role of Non-Neutralizing Antibodies in Protecting against Lassa fever

Graham Cox – 7739345

MMIC 7050 – Microbial Pathogenesis

Advisors – Drs. Michael Mulvey and Amrita Bharat

Assigned: Tuesday, November 6, 2018

Due: Wednesday, November 28, 2018

Background:

The term viral hemorrhagic fever is one that has become very familiar with many over the past number of years, thanks in large part to the recent Ebola outbreak that ravaged areas of western Africa in 2014 and 2015. These diseases, known as VHFs, are containment-level 4 pathogens and are known to be caused by four viral families: Arenaviridae, Bunyaviridae, Filoviridae, and Flaviviridae. These viruses are limited to certain areas of the world, however host distribution is in some cases global and so acquisition of virus particles is possible within or outside its normal range1. The viruses are of the single-stranded ribonucleic acid (ssRNA) type with very short and minimal genomes usually consisting of genes for an RNA polymerase, nucleoprotein, and at most a handful of coat proteins. They are enveloped, zoonotic viruses and together have a wide variety of animal hosts including mammals (bats and rodents) and arthropods (ticks and mosquitoes)2.

VHFs are characterized by a wide range of symptoms which include fever, malaise, dry cough and sore throat, pain in the chest and abdominal areas, and moderate-to-severe hemorrhage at multiple body sites (hence the name) caused by atypical coagulation in affected individuals3. Although general bleeding is very often seen in these cases, it does not usually result in a significant enough amount of blood lost to be considered dangerous4. Other symptoms can also include vascular permeability leading to a loss of plasma from the blood and varying degrees of shock2. Most of these manifestations are in fact results of the hyperactivity of the host’s innate immune system5. Many exact mechanisms are unknown but it is proposed that viral infection of macrophages and monocytes result in a vast increase in cytokines that lead to tissue inflammation which after a prolonged period lead to tissue damage. Dendritic cells are also affected, and along with compromised macrophages severely hamper the body’s ability to mount an effective humoral response, as they are antigen-presenting cells (APCs)3.

There are currently no vaccines in place for many VHFs, an issue which was all too relevant during the 2014-15 West Africa Ebola outbreak2. Now Lassa virus (LASV), a VHF belonging to the Arenavirus family and the causative agent of Lassa fever (LF), is further exacerbating this fact. Since early 2018, there has been an ongoing LF outbreak taking place in Nigeria. With 553 confirmed cases and a case fatality rate of 25.9%, it has been devastating to local populations6. LF has an approximate incubation period of 1-3 weeks and it is believed to only be symptomatic in 20% of infected humans7. It can be passed on via contact with contaminated rodent feces and urine (tainted food sees this happen often) as well as through person-to-person contact, although this is rare and mostly occurs in hospital settings where many patients are immunocompromised8. Mastomys natalensis, otherwise known as the common African rat, is the natural reservoir of this disease and is found throughout much of western and sub-Saharan Africa, where the disease is seen most frequently9. Patients that succumb to LF normally present with a very high viremia. This is due to the fact that the virus can enter leukocytes and as previously mentioned above, leading to excessive inflammation owing to the release of a number of cytokines7.

Lassa Virus Study Re view:

The scientific article being summarized here is titled “Non-neutralizing antibodies elicited by recombinant Lassa–Rabies vaccine are critical for protection against Lassa fever” and is authored by Tiago Abreu-Mota et al. (2018). The researchers conduct multiple experiments to try and determine which part(s) of the immune system are activated when presented with various recombinant virus vectors that have LASV glycoprotein-complex (GPC) incorporated into their genomes with the aims of trying to test whether or not an effective vaccine against LASV can be produced, and to what extent it is useful10. GPC, which is a protein trimer composed of proteins GP1, GP2, and SSP, was used in the recombinant disease vectors due to the fact that it is a LASV surface protein and it is found to be expressed on the outer membrane of LASV-infected host cells11. Viral entry into host cells is also facilitated by the GPC of LASV12. Various mice and Guinea Pigs were used for this study’s experiments due to their similarities with humans since they are both mammals, and also due to the fact that they are excellent models of diseases in humans, with regards to observed symptoms and response of their immune systems13.

In previous studies mentioned in the article’s introduction on the topic of LF vaccination in animal models, they note how there have been multiple, mixed conclusions drawn with various degrees of confounding results regarding immune system response to LASV. Some have stated very clearly that no humoral response is present in animals who have been given recombinant or reassortment-based viral vectors, and that cell-mediated immunity is the effective branch of the immune system acting to fight off the infection and that it can successfully do so14,15. However, contradictory conclusions have also been made from several studies saying that humoral immunity has been observed to prevent death and even disease entirely. This statement results from experiments in which serum from LF survivors was given to other animals with LF and was effective in preventing the disease normally expected to follow infection16,17. The roles of antibodies has also been debated with regards to LF. A recent study done by Cross et al. (2016) utilizing Guinea pigs showed they were protected from LF after being administered human monoclonal antibodies that (in vitro) specifically targeted GPC allowing for neutralization of LASV18. Generally, of those that live through LF few are found to possess neutralizing antibodies (NAbs) in sera19. The study done by Cross et al. (2016) did not perform any experiments in regards to non-neutralizing antibodies (non-NAbs) that also had specific interactions with LASV GPC, so their role(s) in LF are not known to us. Non-NAbs are however known to allow for effector cell activation. The effector cells in this case are immune cells, and the means by which they are activated, through binding the Fcγ region of the antibody, are referred to as antibody-dependent cell-mediated phagocytosis (ADCP; leads to destruction of virus/compromised host cell by engulfment) and antibody-dependent cell-mediated cytotoxicity (ADCC; target cell is lysed via secretion of enzymes, reactive oxygen species, etc.)20.

The primary objective of the research by Abreu-Mota and colleagues was to determine the roles of cellular and humoral immunities in LF by attaining a better understanding of the role of non-NAbs. This was to be done through the use of various vectors carrying the LASV GPC gene, which would then be put into animal models that would be observed and tested for antibody types and concentrations

In order to begin performing the various experiments that would enable the team to do research, the viral disease vectors had to be constructed. LASSARAB was the name given to the main vector used throughout their study, and is a rhabdoviral construct based off of a codon-optimized BNSP333 strain whereby the arginine at position 333 now has a glutamate in the its genome21. This acts to improve its safety in the animals so that LASV GPC (which is inserted into the genome between the BsiWI and NheI restriction sites) can be studied more effectively (Figure 1). LASSARAB-ΔG was also made by removing the RABV G protein gene from the newly altered genome. Two control vectors were also constructed. The first recombinant vector utilized a vesicular stomatitis virus and would be made to express LASV GPC as well (rVSV-GPC). The second was LASSARAB with an Ebola glycoprotein (similar to LASV GPC) and was deemed FILORAB122.

Once vectors were constructed, it was first tested by Abreu-Mota et al. if the LASV GPC would still be expressed on the surface of cells, even though it is not being carried on its native viral vector (LASV). Vero cells (ATCC®CCL81™) were given immunofluorescent antibodies that correspond to LASV GPC and RABV G were used to detect surface expression after they had been infected with two different multiplicities of infection (MOIs) of vectors A through D from Figure 1 (Figure 2a, b). Results were as expected, with LASSARAB showing both stains, LASSARAB-ΔG and rVSV-GPC only showing stain corresponding to LASV GPC, and FILORAB1 only to RABV G. Fig 2c show the results of a growth curve where LASSARAB-ΔG showed slightly more growth from 48 hours on than LASSARAB and FILORAB1 did. They posit that this may be to it lacking RABV G and thus having a shorter overall genome enabling it to be made more efficiently, however this has yet to be studied in detail. LASV GPC incorporation into progeny virions was tested using SDS-PAGE and western blot (fig 2d, e). FILORAB1 was run on SDS-PAGE along with LASSARAB. Their respective proteins were all seen with LASV GP2 being very clear and LASV GP1 a bit less clear since it was assumed to be co-migrating with RABV P, and this was confirmed with the results of the western blot, having a weight of around 50 kDa.

Tested next was whether or not LASSARAB caused LF or Rabies in mice, since an effective vaccine should not cause any disease whatsoever. Mice were given either LASSARAB, LASSARAB-ΔG, FILORAB1 (where FILORAB1 is a negative control) or rVSV-GPC, or were given Phosphate-buffered saline (PBS) as second negative control with the BNSP333 parent vector being used as a positive control. These doses were all given both intranasally (IN) and intraperitoneally (IP) and conditions were examined for a span of four weeks. They saw that on day eight mice were adversely affected by the BNSP333 parent vector (evident by a significant loss of mass) and all mice had died by day 11 (Figure 3a). Meanwhile, mice given LASSARAB,

LASSARAB-ΔG, FILORAB1 were all seen to survive with the exception of one mouse from the LASSARAB-ΔG IN group who died suddenly without presentation of symptoms (ruffled fur, back being hunched, etc.). This may have been due to stress among other factors. Also, two of the rVSV-GPC IN mice died with another coming close but recovering from its symptoms of LF. Intracranial (IC) injection into BALB/c and adult severe combined immunodeficiency (SCID) mice (Figure 3b). This method, which would allow the disease (if any) to progress the most rapidly and be the most severe, as it would be highly concentrated near the brain of the animal. However, an increase in pathogenicity to towards the mice was not seen as every single mouse survived. The notably more sensitive Swiss Webster suckling mice23 were also given doses of LASSARAB (in various concentrations) or BNSP333 (Figure 3c) via IN injection to further determine the safety of the vaccine. All of these mice began exhibiting symptoms by day 7 and were dead within 12 days of infection.

The LASSARAB prospective vaccine was to be tested in live-attenuated form and an inactivated form. The live-attenuated virus is a weakened form and thus is still able to replicate, albeit very ineffectively24. Since Abs are the basis of this project, the Abreu-Mota and colleagues attempted to test whether the live vaccine induced the formation of LASV GPC-specific IgGs. Mice (C57BL/6) were given doses of the following vectors: live-attenuated (replication-competant) LASSARAB (ie. rc-LASSARAB), rc-LASSARAB-ΔG, rc-FILORAB1, and some were given rVSV-GPC. These doses were all administered intramuscularly (IM) and samples were taken biweekly, three times (spanning 42 days). By the end period, rc-LASSARAB and rc-FILORAB1 infected mice were both found to have high amounts of RABV G-binding Igs while the other two were not, which hold since their vectors did not possess RABV G. They were however found to have reacted in accordance with the LASV GPC gene present on each of them. Rc-LASSARAB-ΔG mice were shown to elicit low concentrations of GPC-specific IgG, while an immune response specific to LASV GPC was observed in rVSV-GPC mice (Figure 4c). They also examined if inactivated LASSARAB had the ability to induce IG production. Again, IM injections were used to administer doses of LASSARAB and FILORAB1 to C57BL/6 mice, but these viruses had been inactivated with 10 µg of β-propiolactone (BPL) and so were unable to undergo replication. The two vectors were tested both in standard PBS, and also with in combination with an adjuvant that includes a TLR4 receptor agonist (GLA) in a stable emulsion (SE). Past studies have shown that in animals systems where FILORAB1 or Influenza is present, GLA+SE is effective at improving immune responses and antibody production22,25. Blood was taken from the mice three times (Figure 4a) and it there was found to be a statistically significant amount more IgG in the LASSARAB infected mice (Figure 4b). These antibody titres were then subtyped and it was found that much more were IgG2c than were IgG1. As seen in Figure 4d and 4e, LASSARAB+GLA+SE was able to induce much more IgG2c, which indicates effectiveness at combatting viral pathogens.

Whether or not NAbs were being produced in LASSARAB infected mice was then tested by the group. They utilized a rVSV pseudovirus (ppVSV) for this. The ppVSV did not possess RABV G but was made to express the reporters NanoLuc and eGFP. Significantly high RABV G NAbs were detected when pVSV was pseudotyped with RABV G, as expected. Both inactivated and live-

attenuated LASSARAB infected mouse serum exhibited this result. A WHO standard was also used to show that LASSARAB gave protection for RABV and with all test groups resulting in values a large amount more than the desired value of 0.5 IU/mL, it was shown yet again that LASSARAB is very able to confer immunity towards RABV. LASV GPC Igs were not detected when pVSV were pseudotyped with LASV GPC, therefore no NAbs were induced by LASSARAB.

Following from previous results, since LASSARAB and LASSARAB+GLA+SE were demonstrated to be quite effective in inducing a humoral response (with IgG2c in particular) in mice, similar experiments were carried out in Hartley guinea pigs as a more advanced rodent model. 60 guinea pigs were divided evenly into six groups and were given treatments from 1-3 doses of LASSARAB+GLA+SE, or either LASSARAB, rVSV-GPC, or RabAvert (a pre- and post-exposure prophylaxis Rabies vaccine26). After being exposed the guinea pig-adapted strain of LASV in the following 2 months, significant immunity was seen for the rodents that had seen rVSV-GPC and the 3x exposure of LASSARAB+GLA+SE (Figure 6b, c). The NAb amounts were found to be quite variable when looking at all six of the groups and did not seem to change at all when comparing guinea pigs that had died or lived (Figure 6e), therefore they concluded that NAbs do not significantly impact the outcome of LASV infection. However when total Abs were analyzed, non-NAbs were found to be in much higher amounts pre- and post-challenge when compared to models immunized with RabAvert. This finding led them to conclude that non-NAbs that are specific for LASV GPC confer LF protection.

These non-NAbs were then examined with regards to how exactly immunity to LF was granted, with ADCC and ADCP being complimentary mechanisms under investigation. To examine this, stable cells made to express LASV GPC on their outer membrane acted as the target (infected) cells while C57BL/6 NK cells acted as the effector cells. Sera from C57BL/6 mice (given two doses of either LASSARAB+GLA+SE or FILORAB1+GLA+SE) were then used as media for the target and effector cells, which were incubated in the sera at multiple different ratios (with FILORAB1+GLA+SE being the control serum). It was found that having LASSARAB in serum allowed for the NK cells to be much for effective in killing target cells, regardless of ratio of target to effector cell (E:T) (Figure 7a). This test was also replicated using macrophages as the effector cells and it was found that the macrophages did indeed also act heavily on the LASSARB sera, killing and also internalizing target cells by way of the Fcγ-R receptor. This was tested by incubating the macrophages with α-Fcγ-RIII mAb and observing no significant levels of target cell phagocytosis (Figure 7c, d).

Throughout the course of this study, Abreu-Mota et al. we able to communicate some very strong conclusions to readers. LASSARAB and LASSARABΔ that were chemically inactivated proved to be much more effective at inducing humoral immunity that resulted in LASV GPC-specific non-NAbs than did the live-attenuated forms of the viruses. In combination with GLA+SE, the outcome was an abundance of IgG2c antibodies, which are more effective than IgG1 with regards to fighting off viral pathogens. Non-NAbs were also found to be correlated with ADCC and ADCP activity, whereby if cells are compromised (and are therefore expressing

LASV GPC) the non-NAbs aid in the widespread and rapid destruction of these cells via the extracellular killing of NK cells and macrophages, and also through the phagocytic action of macrophages via the Fcγ-R on the immune cells themselves. Being an inactivated virus, LASSARAB can be safely used in sizeable quantities on more sensitive individuals such as elderly, very young children, and the immunocompromised population. It can also be used to grant resistance to Rabies, an ongoing challenge in many African countries.

Figures and Tables:

Figure 1. All experimental and control vectors used in the study by Abreu-Mota et al. (2018).

Figure 2. Results indicating both LASV GPC and RABV G are both expressed on the surface of infected cells and virions.

Figure 3. Results indicating the virulence (in mice) of the multitude of recombinant vectors used throughout the experiment.

Figure 4. Antibody responses elicited by C57BL/6 mice after being IM injected with various viruses with and without the GLA+SE adjuvant.

Figure 5. Visualization using NanoLuc and eGFP reporters of antibody production by pVSV pseudotyped with either RAVB G or LASV GPC.

Figure 6. LF resistance observed in guinea pig populations that had been immunized with various doses of LASSARAB+GLA+SE, and doses of LASSARAB, rVSV-GPC, and RabAvert.

Figure 7. Experiments showing that ADCC and ADCP are promoted by LASV GPC-specific non-NAbs.

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