Antibody-mediated Enhancement and Neutralization in the Context of Emerging Flavi- and Hantaviruses

Recent outbreaks of emerging viruses underscore the necessity of society-level commitments to research and development. In particular, research into how these threats function at the molecular level is imperative, and leveraging these advances towards the development of countermeasures is the obvious next step. If global trends in climate change, economic growth, and population expansion continue, human exposure will only increase. The 2015-2016 epidemic of Zika virus (ZIKV) is a key example of this phenomenon. The rising frequency and severity of hantavirus outbreaks in Chile and Argentina can also be partially attributed to these forces. Of particular interest is the emergent phenomenon of antibody-dependent enhancement (ADE). In ADE, cross-reactive immunoglobulin G (IgG) from a prior infection bind a related virus, fail to neutralize, and instead provide a mode of attachment allowing the novel virus to infect vulnerable cells through Fc-FcR interactions. During the 2015-2016 outbreak, ADE of dengue virus (DENV)-specific IgG was hypothesized as a mechanism through which ZIKV achieved a more widespread and severe pathogenicity. We were particularly interested in investigating ZIKV ADE in the context of mouse models of infection. Connecting these different properties in vitro and in vivo may allow us to predict if ADE exacerbates human disease. To that end, we examined the plasma of DENV- and West Nile virus (WNV)-infected blood donors. Compared to controls, DENV- and WNV-infected samples displayed significantly greater reactivity against ZIKV envelope (E) protein in ELISA. This increased binding also translated to dosage-mediated enhancement of ZIKV infectivity when tested in vitro. Stat2-/- mice injected with a pool of the most enhancing DENV+ plasma succumbed to ZIKV-infection at significantly higher rates compared to those injected with pooled control plasma. Mice receiving WNV-infected plasma displayed intermediate survival levels, coinciding with intermediate binding and enhancement data. We were especially interested in how phylogenetics influences ADE, as immunity against several flaviviruses is highly seroprevalent in the populations of multiple European and Asian countries. Tick-borne encephalitis virus (TBEV) presented a unique opportunity to explore these questions, as TBEV is one of the most distantly related flaviviruses to ZIKV. Vaccines against TBEV are also in widespread use throughout Germany, Austria, Switzerland, and Lithuania. When we examined sera from TBEV vaccinees, we found widespread cross-reactivity against ZIKV, but intermediate enhancement of ZIKV infection in vitro and negligible enhancement in vivo. Enhancement, though, is a complicating factor observed only rarely across the whole of single-stranded viral families. ADE is likely driven by conserved epitopes present on the glycoproteins of related viruses, but why and how it occurs in flaviviruses and not elsewhere is yet to be determined. To further characterize properties associated with conserved epitopes, we resolved to examine another viral genus responsible for semi-seasonal outbreaks in rural South America: orthohantaviruses. Conserved and specific epitopes on the envelope glycoproteins (GnGc) of the severely lethal orthohantaviruses, including Andes virus (ANDV), are understudied and could provide evidence for cross-neutralization. To test this, we generated 19 distinct monoclonal antibodies (mAbs) against ANDV GnGc using murine hybridoma technology. In characterizing these mAbs, we observed overall trends in and correlations between specificity, epitope, neutralization, effector function, and protection against ANDV-induced disease in an animal model. These data suggest the existence of a wide array of neutralizing and protective antibody epitopes on hantavirus GnGc with unique properties and mechanisms of action. Overall, the data presented here give definition to the relationship between reactivity against envelope glycoproteins, enhancement, neutralization, and protection in the context of these two emerging viral families. This knowledge will also drive innovation towards therapeutics and vaccines to help combat these imminent viral threats

Antibodies to the Glycoprotein GP2 Subunit Cross-React between Old and New World Arenaviruses

Several viruses in the Arenaviridae family infect humans and cause severe hemorrhagic fevers which lead to high case fatality rates. Due to their pathogenicity and geographic tropisms, these viruses remain very understudied. As a result, an effective vaccine or therapy is urgently needed. Here, we describe efforts to produce cross-reactive monoclonal antibodies that bind to both New and Old World arenaviruses. All of our MAbs seem to be nonneutralizing and nonprotective and target subunit 2 of the glycoprotein. Due to the lack of reagents such as recombinant glycoproteins and antibodies for rapid detection assays, our MAbs could be beneficial as analytic and diagnostic tools.Arenaviruses pose a major public health threat and cause numerous infections in humans each year. Although most viruses belonging to this family do not cause disease in humans, some arenaviruses, such as Lassa virus and Machupo virus, are the etiological agents of lethal hemorrhagic fevers. The absence of a currently licensed vaccine and the highly pathogenic nature of these viruses both make the necessity of developing viable vaccines and therapeutics all the more urgent. Arenaviruses have a single glycoprotein on the surface of virions, the glycoprotein complex (GPC), and this protein can be used as a target for vaccine development. Here, we describe immunization strategies to generate monoclonal antibodies (MAbs) that cross-react between the glycoprotein complexes of both Old World and New World arenaviruses. Several monoclonal antibodies isolated from immunized mice were highly cross-reactive, binding a range of Old World arenavirus glycoproteins, including that of Lassa virus. One such monoclonal antibody, KL-AV-2A1, bound to GPCs of both New World and Old World viruses, including Lassa and Machupo viruses. These cross-reactive antibodies bound to epitopes present on the glycoprotein 2 subunit of the glycoprotein complex, which is relatively conserved among arenaviruses. Monoclonal antibodies binding to these epitopes, however, did not inhibit viral entry as they failed to neutralize a replication-competent vesicular stomatitis virus pseudotyped with the Lassa virus glycoprotein complex in vitro. In addition, no protection from virus challenge was observed in in vivo mouse models. Even so, these monoclonal antibodies might still prove to be useful in the development of clinical and diagnostic assays

Neutralizing Monoclonal Antibodies against the Gn and the Gc of the Andes Virus Glycoprotein Spike Complex Protect from Virus Challenge in a Preclinical Hamster Model

Infections with New World hantaviruses are associated with high case fatality rates, and no specific vaccine or treatment options exist. Furthermore, the biology of the hantaviral GnGc complex, its antigenicity, and its fusion machinery are poorly understood. Protective monoclonal antibodies against GnGc have the potential to be developed into therapeutics against hantaviral disease and are also great tools to elucidate the biology of the glycoprotein complex.Hantaviruses are the etiological agent of hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS). The latter is associated with case fatality rates ranging from 30% to 50%. HCPS cases are rare, with approximately 300 recorded annually in the Americas. Recently, an HCPS outbreak of unprecedented size has been occurring in and around Epuyén, in the southwestern Argentinian state of Chubut. Since November of 2018, at least 29 cases have been laboratory confirmed, and human-to-human transmission is suspected. Despite posing a significant threat to public health, no treatment or vaccine is available for hantaviral disease. Here, we describe an effort to identify, characterize, and develop neutralizing and protective antibodies against the glycoprotein complex (Gn and Gc) of Andes virus (ANDV), the causative agent of the Epuyén outbreak. Using murine hybridoma technology, we generated 19 distinct monoclonal antibodies (MAbs) against ANDV GnGc. When tested for neutralization against a recombinant vesicular stomatitis virus expressing the Andes glycoprotein (GP) (VSV-ANDV), 12 MAbs showed potent neutralization and 8 showed activity in an antibody-dependent cellular cytotoxicity reporter assay. Escape mutant analysis revealed that neutralizing MAbs targeted both the Gn and the Gc. Four MAbs that bound different epitopes were selected for preclinical studies and were found to be 100% protective against lethality in a Syrian hamster model of ANDV infection. These data suggest the existence of a wide array of neutralizing antibody epitopes on hantavirus GnGc with unique properties and mechanisms of action

Tick-Borne Encephalitis Virus Vaccine-Induced Human Antibodies Mediate Negligible Enhancement of Zika Virus Infection InVitro and in a Mouse Model

ABSTRACT Recent reports in the scientific literature have suggested that anti-dengue virus (DENV) and anti-West Nile virus (WNV) immunity exacerbates Zika virus (ZIKV) pathogenesis in vitro and in vivo in mouse models. Large populations of immune individuals exist for a related flavivirus (tick-borne encephalitis virus [TBEV]), due to large-scale vaccination campaigns and endemic circulation throughout most of northern Europe and the southern Russian Federation. As a result, the question of whether anti-TBEV immunity can affect Zika virus pathogenesis is a pertinent one. For this study, we obtained 50 serum samples from individuals vaccinated with the TBEV vaccine FSME-IMMUN (Central European/Neudörfl strain) and evaluated their enhancement capacity in vitro using K562 human myeloid cells expressing CD32 and in vivo using a mouse model of ZIKV pathogenesis. Among the 50 TBEV vaccinee samples evaluated, 29 had detectable reactivity against ZIKV envelope (E) protein by enzyme-linked immunosorbent assay (ELISA), and 36 showed enhancement of ZIKV infection in vitro. A pool of the most highly reacting and enhanced samples resulted in no significant change in the morbidity/mortality of ZIKV disease in immunocompromised Stat2−/− mice. Our results suggest that humoral immunity against TBEV is unlikely to enhance Zika virus pathogenesis in humans. No clinical reports indicating that TBEV vaccinees experiencing enhanced ZIKV disease have been published so far, and though the epidemiological data are sparse, our findings suggest that there is little reason for concern. This study also displays a clear relationship between the phylogenetic distance between two flaviviruses and their capacity for pathogenic enhancement. IMPORTANCE The relationship between serial infections of two different serotypes of dengue virus and more severe disease courses is well-documented in the literature, driven by so-called antibody-dependent enhancement (ADE). Recently, studies have shown the possibility of ADE in cells exposed to anti-DENV human plasma and then infected with ZIKV and also in mouse models of ZIKV pathogenesis after passive transfer of anti-DENV human plasma. In this study, we evaluated the extent to which this phenomenon occurs using sera from individuals immunized against tick-borne encephalitis virus (TBEV). This is highly relevant, since large proportions of the European population are vaccinated against TBEV or otherwise seropositive