Humoral immunity to phlebovirus infection

Abstract Phleboviruses are zoonotic pathogens found in parts of Africa, Asia, Europe, and North America and cause disease symptoms ranging from self‐limiting febrile illness to severe disease, including hemorrhagic diathesis, encephalitis, and ocular pathologies. There are currently no approved preventative vaccines against phlebovirus infection or antivirals for the treatment of the disease. Here, we discuss the roles of neutralizing antibodies in phlebovirus infection, the antigenic targets present on the mature polyproteins Gn and Gc, progress in vaccine development, and the prospects of identifying conserved neutralizing epitopes across multiple phleboviruses. Further research in this area will pave the way for the rational design of pan‐phlebovirus vaccines that will protect against both known phleboviruses but also newly emerging phleboviruses that may have pandemic potential.


BIOMEDICAL BURDEN OF PHLEBOVIRUSES
Phleboviruses are a genetically diverse group of viruses from the family Phenuiviridae, order Bunyavirales.A well-known phlebovirus member is Rift Valley Fever Virus (RVFV), an arbovirus that is the causative agent of Rift Valley Fever (RVF).Endemic to Africa 1 and more recently the Arabian Peninsula, 2 it can cause recurrent epidemics, with human disease ranging from mild self-limiting febrile illness to severe disease, including hemorrhagic diatheses, encephalitis, and ocular pathologies. 3,4Ruminants are the major reservoirs of RVFV, and infection in livestock leads to high rates of neonatal mortality, resulting in major losses to the livestock industry in endemic areas. 5RVFV is transmitted to humans through bites of infected mosquitos or through contact with the blood or organs of infected animals. 6ere are 10 other reported species within the phlebovirus family, of which some have been reported in repeated zoonotic events. 7scana virus (TOSV) is a phlebovirus isolated from two species of sandfly in Italy. 8Infections mostly occur in Mediterranean countries in the summer months 9 and can cause acute neurological diseases.
Heartland virus (HRTV) is the first pathogenic phlebovirus observed in the United States 10,11 and is most closely related to severe fever with thrombocytopenia syndrome virus (SFTSV).SFTSV, which was first reported in China in 2007, 12 was originally classified as a phlebovirus but was recently reclassified in the genus Bandavirus of the Phenuiviridae family, order Bunyavirales 13 and officially named Dabie bandavirus.Due to the previous classification of SFTSV as a phlebovirus and the literature comparing SFTSV and RVFV structures and antibody responses, [14][15][16] SFTSV has been included in this review.
SFTSV is characterized by a sudden onset fever, respiratory or gastrointestinal symptoms, and progression to hemorrhage and multiorgan failure, 17 and it has an average case fatality rate of 12%. 18ansmission of SFTSV is predominantly associated with arthropod bites but person-to-person transmission through blood contact has also been reported. 18,19High SFTSV seroprevalence in domestic animals has been reported in endemic regions, demonstrating a number of different vertebrate hosts. 20,21 I G U R E 1 RVFV Gn-Gc assembly.(A) Schematic showing the arrangement of RVFV Gn (pink) and Gc (dark gray) on the virion surface.(B) Gn-Gc complex assembly of RVFV (image created in ChimeraX using EMB-4197).31 Phleboviruses are a significant zoonotic and pandemic threat to both human and animal populations due to the global presence of both the hosts (including livestock and domestic animals) and the arthropod vectors (including mosquito, tick, and sandfly).Indeed, the World Health Organization has reported nine outbreaks of RVFV between 2000 and 2016, infecting over 4600 people and leading to 957 deaths.6 However, there are currently no approved vaccines for the prevention of phlebovirus infection or antivirals for the treatment of the disease, although several vaccine approaches [22][23][24][25] and antivirals 26,27 are currently being explored.

PHLEBOVIRUS STRUCTURE
Phleboviruses are enveloped single-stranded negative-sense RNA viruses, and their genomes are divided into three segments: S, M, and L. The S segment encodes the nucleoprotein and a nonstructural protein (NSs), the M segment encodes the structural glycoproteins, and the L segment encodes the RNA-dependent RNA polymerase.
9][30][31] Gn and Gc are essential for viral attachment and host cell entry.In the case of RVFV, host-cell entry begins with the interaction between the high-mannose glycans on Gn and the C-type lectin DC-SIGN. 32This interaction initiates caveolae-mediated endocytosis. 33Gc harbors a class II fusion protein and facilitates membrane fusion utilizing a pH-dependent mechanism in the acidic environment of an endosomal compartment. 31ystal structures of the Gn of RVFV 14,15,31 and SFTSV 16 reveal a three-domain architecture consisting of (i) domain A, an α-helical/βstranded N-terminal domain; (ii) a β-ribbon domain; and (iii) domain B (Figure 2A).However, although the Gn molecules from RVFV and SFTSV display the same overall fold (Figure 2B,C), there are differences in the arrangement of subdomains, demonstrating the structural variation between Phenuiviridae family members. 16In contrast to Gn, the Gc structure is relatively conserved between RVFV, 34 SFTSV, 35 and HRTV. 36Gc has three domains (termed I-III) that are composed of β-sheets (Figure 3A,B).Domain II contains the fusion loops.Localization of the crystalized fragments of RVFV Gn and Gc in purified RVFV cryo-EM-based single particle reconstructions shows that the N-terminal domain of Gn is membrane-distal to the viral envelope and shields both Gc and its hydrophobic fusion loop, preventing premature fusion. 31Further structural studies comparing the higher-order ultrastructures of SFTSV and HRTV are required to determine whether this arrangement is conserved across the phlebovirus family. 37

HUMORAL IMMUNE RESPONSE TO PHLEBOVIRUS INFECTION
Gn and Gc are the key targets for neutralizing antibodies elicited following infection and, therefore, the main antigens of interest for vaccine development.Neutralizing antibodies have been identified following RVFV infection in livestock, 1 nonhuman primates, 38 and humans. 39The protective capacity of neutralizing antibodies has been demonstrated through passive transfer of convalescent sera to susceptible animals. 1,38Furthermore, data from vaccine studies have demonstrated that high serum-neutralizing titers are sufficient to provide protection in animal challenge models. 402][43] Immune correlates of protection against RVFV have been studied in mice vaccinated with an RVFV attenuated vaccine (DelNSsRVFV). 40Mice were protected from lethal RVFV challenge with or without depletion of CD4 + and/or CD8 + T cells, or B cells prior to RVFV challenge.However, the passive transfer of sera from immunized mice provided vaccine-naïve mice with complete protection from lethal challenge, confirming the role of neutralizing antibodies in protection.While these studies do not preclude the role that non-neutralizing Gnand/or Gc-specific antibodies might be playing in protection, a study by Cartwright et al. fails to demonstrate the protective capacity of three non-neutralizing monoclonal antibodies (mAbs) compared to neutralizing mAbs. 44However, IgG2a versions of neutralizing mAbs conferred better protection than the corresponding IgG1 version, suggesting that antibody-mediated effector functions may play a role in the protection.

Gn and Gc are the major antigenic determinants and targets for neutralizing antibodies arising following infection. A recent study by
Wright et al. demonstrated by depletion of Gn-or Gc-specific antibodies from sera of RVFV convalescent donors that the majority of the neutralizing activity is directed against Gn and a minor proportion against Gc. 39This is consistent with structural studies showing that Gn shields Gc in the RVFV prefusion conformation. 31ile the immune response to RVFV infection has been the most extensively studied, antibody responses to other phlebovirus similarly reveal Gn and Gc to be the major targets for infection-derived immunity. 45Similar to RVFV, Gn and Gc are targeted by neutralizing antibodies following SFTSV 46 or TOSV infection. 47

ANTIGENIC TARGETS ON PHLEBOVIRUSES AND SFTSV
Isolation and characterization of mAbs from convalescent donors or vaccinated animals provides a platform to characterize sites of immune vulnerability on the Gn and Gc of phleboviruses that can be targeted by mAb therapies and that can inform rational development of phlebovirus vaccines.The majority of studies isolating phlebovirus mAbs have focused on RVFV and SFTSV (summarized in Table 1).Confirming analysis of the neutralizing activity in convalescent sera, neutralizing mAbs targeting both the Gn and Gc of phleboviruses have been isolated and characterized, and their protective capacity determined.Many of the neutralizing and protective mAbs described in Table 1 represent important candidates for the development of antibody-based therapeutics for the prevention and/or treatment of phlebovirus infections.

Neutralizing epitopes on Gn
The majority of phlebovirus-specific neutralizing mAbs reported thus far target Gn, which is consistent with Gn being most highly exposed on the virus surface 35 and the dominance of Gn-specific neutralizing activity in convalescent sera. 39mAbs specific for RVFV Gn have been isolated from both infected and vaccinated humans, as well as from vaccinated animals.
Wang et al. isolated mAbs from an RVFV convalescent donor using antigen-specific B cell sorting with Gn and Gc antigen baits. 15Eight neutralizing Gn-specific mAbs were isolated (R4, R12, R13, R15, R16, R17, R19, and R22), all displaying low levels of somatic hypermutation and all binding to Gn domain A (Figure 2 and Table 1).A study of their mechanism of neutralization showed inhibition of RVFV interaction with Vero cells, suggesting direct inhibition of receptor binding.While all mAbs bound Gn domain A, structural analysis and epitope mapping revealed three overlapping neutralization hotspots within this domain.
Sequence analysis of 108 RVFV strains showed strong conservation of residues within the mAb epitopes, predicting broad activity.
Chapman et al. selected neutralizing mAbs from both RVFV convalescent donors as well as individuals immunized with RVF MP-12 (a live attenuated RVFV vaccine 49 ) through the generation of B-cell lymphoblastoid cell lines. 50In addition to Gn domain A-specific mAbs (RVFV-142, RVFV-268, RVFV-379, RVFV-436, RVFV-426, RVFV-296, and RVFV-401), domain B-specific neutralizing mAbs (RVFV-226, RVFV-381, RVFV-405, and RVFV-429) were also isolated (Figure 2 and Table 1).In contrast to the neutralization mechanism of receptor binding inhibition proposed by Wang et al., 15 Gn-specific mAbs that could inhibit the Gn interaction with cells were unable to completely inhibit binding of the receptor, LRP1, in biolayer interferometry competition experiments, indicating that complete blocking of the Gn-LRP1 interaction is not essential for potent neutralization by this class of mAbs. 51Furthermore, some Gn-reactive mAbs isolated by Chapman The immunodominance of Gn domain B was further exemplified by the isolation of RVFV domain B-reactive mAbs from rabbits immunized with recombinant RVFV Gn (mAb RV-Gn1) 14 and from macaques immunized with recombinant human adenovirus type 4 expressing RVFV Gn and Gc proteins (rHAdV4-GnGcopt) (mAbs 1332F11 and 1331E4), 52 and by isolation of a SFTSV domain B-reactive mAb (MAb 4-5) from a convalescent donor. 16,53Despite RV-Gn1 and MAb 4-5 showing distinct modes of binding and contacting antigenically distinct surfaces, a comparison of the Fab crystal structures in complex with RVFV Gn and SFTSV Gn, respectively, showed their epitopes localize to the same domain.Combined, these mAbs highlight domain B as a common site of immune vulnerability on phlebovirus Gn and, therefore, an important domain for rational vaccine design strategies. 14wever, while RVFV mAb RV-Gn1 was shown to be neutralizing and protective in a pre-exposure RVFV infection mouse model, 14 SFTSV MAb 4-5, which was also neutralizing in vitro, was not protective in an SFTSV pre-exposure lethal challenge study in mice, 54 suggesting different mechanisms of action for domain B-specific mAbs against specific phleboviruses and SFTSV.
No RVFV mAbs have been reported to bind the β-ribbon domain. 15,50However, the epitope of mAb Ab10 isolated from an SFTSV convalescent donor was shown using crosslinking coupled mass spectrometry and alanine scanning mutagenesis to be located to the Gn β-ribbon domain and the stem region of Gn. 54 Although further structural analysis is required to determine the exact molecular inter-actions, the proposed neutralization mechanism is inhibition of the un-shielding of the Gc fusion loop.mAb Ab10 was shown to protect both pre-and post-exposure from a lethal SFTSV challenge dose in a type 1 interferon-deficient mouse model. 54utralizing mAbs (TVB 27, TVB 73, TVB 147, TVB 161, and TVB 164) have been isolated from a TOSV convalescent donor through the immortalization of memory B cells (Table 1). 55The neutralizing epitopes were mapped using pepscan, which revealed epitopes located in the N-terminus (domain A) and near to the transmembrane region.
Further analysis is required to determine the epitope location in TOSV Gn.

Neutralizing epitopes on Gc
In the prefusion state, the Gc fusion loop is shielded by Gn and, therefore, this virally encoded antigen is less exposed to the immune system during infection.Indeed, only a handful of Gc-specific mAbs have been reported thus far (Table 1).RVFV Gc-specific mAb R5 was isolated from an RVFV convalescent donor and was found to be less potently neutralizing than Gn-specific mAbs isolated from the same donor. 15  RVFV-249) lost binding with mutations in domain I of Gc.The protective capacity of these Gc-specific mAbs was not determined.Five SFTSV Gc-reactive scFvs were isolated from a phage library generated from an SFTSV convalescent donor but the neutralization capacity or analysis of their epitopes has not been reported. 54

Quaternary epitopes spanning Gn-Gc
Neutralizing antibodies binding a quaternary epitope spanning multiple glycoproteins have been identified for HIV-1. 56,57Isolation of phlebovirus-neutralizing mAbs targeting quaternary epitopes spanning Gn and Gc would require antigen baits displaying the native Gn/Gc configuration, which are currently not available in recombinant form.In an alternative approach, Chapman et al. selected B cells through screening for neutralization activity in the supernatants of immortalized B cells from an RVF MP-12-vaccinated individual (a live attenuated vaccine).Two mAbs (RVFV-140 and RVFV-144) were able to bind cells transfected with the full M segment but not to cells expressing Gn or Gc alone, indicating binding to a quaternary Gn/Gc epitope (Table 1). 50While the epitope targeting by these mAbs still needs to be defined at the molecular level, a representative mAb from this group was shown to protect in an RVFV pre-and post-exposure mouse model. 50

PROSPECTS FOR DEVELOPMENT OF CROSS-REACTIVE NEUTRALIZING ANTIBODIES
Although mAbs have been identified that show potent neutralization and protection against RVFV or SFTSV, isolation of mAbs with crossneutralizing or cross-protective activity against multiple phleboviruses has thus far proved elusive.This is despite the epitopes of RVFV-and STSFV-specific mAbs being mapped to similar domains on Gn. 14,16,53 Gn, which a high proportion of mAbs target, is the most exposed glycoprotein on the virus surface and as a predominant target for neutralizing antibodies elicited following infection is under the highest level of antigenic selection.Analysis of the absolute rate of nucleotide substitution ratios (dN/dS) for RVFV gene sequences sampled between 1951 and 2010 revealed regions of Gn and Gc to be under the greatest selective pressure. 14When comparing Gn and Gc, Gn was found to have a slightly higher mean dN/dS ratio compared to Gc.However, when Gn was separated into domains, it was found that domain B had the highest dN/dS ratio compared to domain A and the β-ribbon domain, indicative of domain B being subject to the greatest level of antibody selective pressure and thus a dominant target of neutralizing antibodies arising from infection.However, the level of variation in this highly exposed domain will unlikely generate cross-protective neutralizing antibodies.
In contrast, Gc is more conserved due to the shared mechanism of membrane fusion across phleboviruses, 58 and the reduced accessibility to neutralizing antibodies 39 leads to lower levels of antigenic selection sequence diversity. 14,39Therefore, cross-neutralizing epitopes present across multiple phlebovirus species will more likely be identified on Gc.

PHLEBOVIRUS VACCINE DEVELOPMENT
The development of effective vaccines that protect animals and humans from known and newly emerging phleboviruses would have a significant impact on global health as well as the economic burden of animal deaths.As outbreaks of RVFV often precede outbreaks in humans by a few weeks, 22 it is important to develop vaccines that can be used in animal reservoirs as well as humans.A variety of vaccine approaches against RVFV have been explored, 59,60 of which several have been evaluated in humans with the aim of generating a neutralizing antibody response.The RVFV-inactivated vaccine TSI-GSD-200, although having a good safety profile in humans, ∼10% of participants immunized with it did not seroconvert despite the administration of three vaccine doses. 61An RVFV live attenuated vaccine, RVF MP-12, was also safe and effective at generating neutralizing antibodies. 23,24The fact that neutralizing RVFV mAbs from MP-12immunized humans have been isolated supports further development of this vaccine. 50Most recently, a nonreplicating simian adenovirus vectored RVFV vaccine encoding Gn and Gc (ChAdOx1 RVF) has been tested in Phase 1 trials. 25Similar to studies conducted in livestock, 62,63 high titers of RVFV-neutralizing antibodies were observed in 12 of 15 participants following a single vaccination and these levels were maintained during the 3-month follow-up period.Interestingly, higher titers of Gc-reactive IgG were detected than Gn-reactive IgG.ChAdOx1 RVF is a promising vaccine candidate due to the development of a scalable manufacturing process used for the ChAdOx1 nCoV-19 vaccine that has been used in over 180 countries. 64,65ile Phase 1 trials in humans have not yet been conducted for other phleboviruses, several approaches for SFTSV have been explored in animal models with varying degrees of success. 66A DNA vaccine encoding full-length Gn, Gc, N, NS, and RdRp genes of SFTSV generated both neutralizing antibodies and a T cell response that was able to protect from lethal challenge in a ferret challenge model. 67replication-deficient human adenovirus type 5 (rAd5) expressing SFTSV Gn prime and recombinant Gn protein boost induced a potent neutralizing antibody response in mice and nonhuman primates and provided protection against lethal SFTSV challenge in mice.68 Furthermore, a live attenuated virus vaccine consisting of a replicationcompetent rVSV vector expressing SFTSV Gn/Gc was shown to elicit broad neutralizing antibodies against both SFTSV and HRTV in both immunocompetent and immunocompromised mice and provided protection from lethal challenge.69 Therefore, there are several promising vaccine candidates for specific phleboviruses that may be adapted to the development of vaccines against other pathogenic and newly emerging phleboviruses.

CONCLUSIONS AND OUTSTANDING QUESTIONS
Neutralizing antibodies play an important role in virus clearance upon infection and are able to prevent phlebovirus infection in animal challenge models. 1,38,40While neutralizing epitopes have been identified on the surface glycoproteins of RVFV, SFTSV, and TOSV, mAbs with cross-binding or cross-neutralizing activity against multiple phlebovirus families have not been identified.Encouragingly, a number of vaccine approaches, including recombinant Gn, 14 live attenuated vaccines, 23,24 and viral-vectored vaccines, 25 readily generate high titers of Gn-and Gc-specific IgG that, unlike HIV-1 broadly neutralizing antibodies, 70 do not require extensive somatic hypermutation for neutralization activity.Therefore, knowledge of the M segment sequence of a newly emerging phlebovirus species with pandemic potential would rapidly pave the way to vaccine development using the strategies outlined above.However, the ultimate goal in controlling phlebovirus infections is the development of vaccines that provide broad protection against both known phleboviruses that are causing ongoing zoonosis events, as well as newly emerging phleboviruses with unknown pandemic potential.Through a more complete knowledge of the sites of immune vulnerability on the Gn and Gc surface glycoproteins, conserved antigenic regions can be identified that can be targeted through reverse vaccinology and rational vaccine design.From a global perspective, the challenge remains to identify cost-effective vaccine formulations for use in animal populations, which will be critical for reducing the phlebovirus animal reservoir and the subsequent spill-over into human populations.
Convalescentsera from individuals recovered from SFTSV was shown to protect from lethal infection in a mouse model when administered after SFTSV exposure.48

F I G U R E 2
Phlebovirus Gn structure.(A) Schematic showing the domain organization for RVFV and SFTSV Gn.Structurally identified domains are colored (domain A: red; domain B: blue; β-ribbon domain: pink).(B) Structure of RVFV Gn (PDB: 6F8P 31 ), with structural domains colored as in panel A. (C) Structure of SFTSV Gn (PDB: 5Y10 16 ).
et al. were shown to inhibit fusion through an indirect mode whereby mAb binding to Gn prevented subsequent exposure of the viral fusion loop. 50Although competition ELISAs revealed a single overlapping epitope on domain A and domain B, alanine mutagenesis revealed that the F I G U R E 3 Phlebovirus Gc structure.(A) Domain organization of RVFV Gc.Structurally identified domains are colored (domain I: blue; domain II: yellow; domain III: red).(B) Structure of RVFV Gc (PBD: 4HJ1 34 ), with structural domains colored as in panel A. binding of mAbs was dependent on different sets of amino acids on either domain A or domain B. Domain A-specific mAbs relied heavily on amino acids present in two Gn pockets (residues 164-186 and 270-294), whereas domain B-specific mAbs were more diverse in their recognition, with some mAbs reliant on domain A for binding as well.
mAb R5 did not protect mice from lethal challenge in either a pre-or post-exposure infection model.In a study by Chapman et al., they isolated50 five RVFV Gc-specific mAbs that formed three binding competition groups.Although these mAbs demonstrated neutralizing activity, some residual infection was seen in the in vitro assay.A mutagenesis screening approach showed three of the mAbs (belonging to Group B: RVFV-326, RVFV-121, and RVFV-250) lost binding with mutations in the Gc domain II region adjacent to the fusion loop; a further mAb (belonging to Group C: RVFV-128) lost binding with TA B L E 1 Summary of phlebovirus-specific monoclonal antibodies isolated from convalescent or vaccinated human donors or vaccinated animals.