Neutralization Sensitivity Research Articles

Hypervariable region 1 and N-linked glycans of hepatitis C regulate virion neutralization by modulating envelope conformations

About two million new cases of hepatitis C virus (HCV) infections annually underscore the urgent need for a vaccine. However, this effort has proven challenging because HCV evades neutralizing antibodies (NAbs) through molecular features of viral envelope glycoprotein E2, including hypervariable region 1 (HVR1) and N-linked glycans. Here, we observe large variation in the effects of removing individual E2 glycans across HCV strains H77(genotype 1a), J6(2a), and S52(3a) in Huh7.5 cell infections. Also, glycan-mediated effects on neutralization sensitivity were completely HVR1-dependent, and neutralization data were consistent with indirect protection of epitopes, as opposed to direct steric shielding. Indeed, the effect of removing each glycan was similar both in type (protective or sensitizing) and relative strength across four nonoverlapping neutralization epitopes. Temperature-dependent neutralization (e.g., virus breathing) assays indicated that both HVR1 and protective glycans stabilized a closed, difficult to neutralize, envelope conformation. This stabilizing effect was hierarchical as removal of HVR1 fully destabilized closed conformations, irrespective of glycan status, consistent with increased instability at acidic pH and high temperatures. Finally, we observed a strong correlation between neutralization sensitivity and scavenger receptor BI dependency during viral entry. In conclusion, our study indicates that HVR1 and glycans regulate HCV neutralization by shifting the equilibrium between open and closed envelope conformations. This regulation appears tightly linked with scavenger receptor BI dependency, suggesting a role of this receptor in transitions from closed to open conformations during entry. This importance of structural dynamics of HCV envelope glycoproteins has critical implications for vaccine development and suggests that similar phenomena could contribute to immune evasion of other viruses.

Prediction of VRC01 neutralization sensitivity by HIV-1 gp160 sequence features.

The broadly neutralizing antibody (bnAb) VRC01 is being evaluated for its efficacy to prevent HIV-1 infection in the Antibody Mediated Prevention (AMP) trials. A secondary objective of AMP utilizes sieve analysis to investigate how VRC01 prevention efficacy (PE) varies with HIV-1 envelope (Env) amino acid (AA) sequence features. An exhaustive analysis that tests how PE depends on every AA feature with sufficient variation would have low statistical power. To design an adequately powered primary sieve analysis for AMP, we modeled VRC01 neutralization as a function of Env AA sequence features of 611 HIV-1 gp160 pseudoviruses from the CATNAP database, with objectives: (1) to develop models that best predict the neutralization readouts; and (2) to rank AA features by their predictive importance with classification and regression methods. The dataset was split in half, and machine learning algorithms were applied to each half, each analyzed separately using cross-validation and hold-out validation. We selected Super Learner, a nonparametric ensemble-based cross-validated learning method, for advancement to the primary sieve analysis. This method predicted the dichotomous resistance outcome of whether the IC50 neutralization titer of VRC01 for a given Env pseudovirus is right-censored (indicating resistance) with an average validated AUC of 0.868 across the two hold-out datasets. Quantitative log IC50 was predicted with an average validated R2 of 0.355. Features predicting neutralization sensitivity or resistance included 26 surface-accessible residues in the VRC01 and CD4 binding footprints, the length of gp120, the length of Env, the number of cysteines in gp120, the number of cysteines in Env, and 4 potential N-linked glycosylation sites; the top features will be advanced to the primary sieve analysis. This modeling framework may also inform the study of VRC01 in the treatment of HIV-infected persons.

Characterization of Mouse Monoclonal Antibodies Against the HA of A(H7N9) Influenza Virus.

Many cases of human infection with the H7N9 virus have been detected in China since 2013. H7N9 viruses are maintained in chickens and are transmitted to humans at live bird markets. During circulation in birds, H7N9 viruses have accumulated amino acid substitutions in their hemagglutinin (HA), which resulted in an antigenically change in the recent H7N9 viruses. Here, we characterized 46 mouse monoclonal antibodies against the HA of the prototype strain. 16 H7-HA-specific monoclonal antibodies (mAbs) possessed hemagglutination inhibition (HI) and neutralization activities by recognizing the major antigenic site A; four other H7-HA-specific clones also showed HI and neutralizing activities via recognition of the major antigenic sites A and D; seven mAbs that reacted with several HA subtypes and possibly recognized the HA stem partially protected mice from lethal infection with prototype H7N9 virus; and the remaining 19 mAbs had neither HI nor neutralization activity. All human H7N9 viruses tested showed a similar neutralization sensitivity to the first group of 16 mAbs, whereas human H7N9 viruses isolated in 2016–2017 were not neutralized by a second group of 4 mAbs. These results suggest that amino acid substitutions at the epitope of the second mAb group appear to be involved in the antigenic drift of the H7N9 viruses. Further analysis is required to fully understand the antigenic change in H7N9 viruses.

Hepatitis C Virus Escape Studies of Human Antibody AR3A Reveal a High Barrier to Resistance and Novel Insights on Viral Antibody Evasion Mechanisms

Yearly, ∼2 million people become hepatitis C virus (HCV) infected, resulting in an elevated lifetime risk for severe liver-related chronic illnesses. Characterizing epitopes of broadly neutralizing antibodies (NAbs), such as AR3A, is critical to guide vaccine development. Previously identified alanine substitutions that can reduce AR3A binding to expressed H77 envelope were introduced into chimeric cell culture-infectious HCV recombinants (HCVcc) H77(core-NS2)/JFH1. Substitutions G523A, G530A, and D535A greatly reduced fitness, and S424A, P525A, and N540A, although viable, conferred only low-level AR3A resistance. Using highly NAb-sensitive hypervariable region 1 (HVR1)-deleted HCVcc, H77/JFH1ΔHVR1 and J6(core-NS2)/JFH1ΔHVR1, we previously reported a low barrier to developing AR5A NAb resistance substitutions. Here, we cultured Huh7.5 cells infected with H77/JFH1, H77/JFH1ΔHVR1, or J6/JFH1ΔHVR1 with AR3A. We identified the resistance envelope substitutions M345T in H77/JFH1, L438S and F442Y in H77/JFH1ΔHVR1, and D431G in J6/JFH1ΔHVR1 M345T increased infectivity and conferred low-level AR3A resistance to H77/JFH1 but not H77/JFH1ΔHVR1 L438S and F442Y conferred high-level AR3A resistance to H77/JFH1ΔHVR1 but abrogated the infectivity of H77/JFH1. D431G conferred AR3A resistance to J6/JFH1ΔHVR1 but not J6/JFH1. This was possibly because D431G conferred broadly increased neutralization sensitivity to J6/JFH1D431G but not J6/JFH1ΔHVR1/D431G while decreasing scavenger receptor class B type I coreceptor dependency. Common substitutions at positions 431 and 442 did not confer high-level resistance in other genotype 2a recombinants [JFH1 or T9(core-NS2)/JFH1]. Although the data indicate that AR3A has a high barrier to resistance, our approach permitted identification of low-level resistance substitutions. Also, the HVR1-dependent effects on AR3A resistance substitutions suggest a complex role of HVR1 in virus escape and receptor usage, with important implications for HCV vaccine development.IMPORTANCE Hepatitis C virus (HCV) is a leading cause of liver-related mortality, and limited treatment accessibility makes vaccine development a high priority. The vaccine-relevant cross-genotype-reactive antibody AR3A has shown high potency, but the ability of the virus to rapidly escape by mutating the AR3A epitope (barrier to resistance) remains unexplored. Here, we succeeded in inducing only low-level AR3A resistance, indicating a higher barrier to resistance than what we have previously reported for AR5A. Furthermore, we identify AR3A resistance substitutions that have hypervariable region 1 (HVR1)-dependent effects on HCV viability and on broad neutralization sensitivity. One of these substitutions increased envelope breathing and decreased scavenger receptor class B type I HCV coreceptor dependency, both in an HVR1-dependent fashion. Thus, we identify novel AR3A-specific resistance substitutions and the role of HVR1 in protecting HCV from AR3-targeting antibodies. These viral escape mechanisms should be taken into consideration in future HCV vaccine development.

Concomitant Enhancement of HIV-1 Replication Potential and Neutralization-Resistance in Concert With Three Adaptive Mutations in Env V1/C2/C4 Domains.

HIV-1 Env protein functions in the entry process and is the target of neutralizing antibodies. Its intrinsically high mutation rate is certainly one of driving forces for persistence/survival in hosts. For optimal replication in various environments, HIV-1 Env must continue to adapt and evolve through balancing sometimes incompatible function, replication fitness, and neutralization sensitivity. We have previously reported that adapted viruses emerge in repeated and prolonged cultures of cells originally infected with a macaque-tropic HIV-1NL4-3 derivative. We have also shown that the adapted viral clones exhibit enhanced growth potentials both in macaque PBMCs and individuals, and that three single-amino acid mutations are present in their Env V1/C2/C4 domains. In this study, we investigated how lab-adapted and highly neutralization-sensitive HIV-1NL4-3 adapts its Env to macaque cells with strongly replication-restrictive nature for HIV-1. While a single and two mutations gave a significantly enhanced replication phenotype in a macaque cell line and also in human cell lines that stably express either human CD4 or macaque CD4, the virus simultaneously carrying the three adaptive mutations always grew best. Entry kinetics of parental and triple mutant viruses were similar, whereas the mutant was significantly more readily inhibited for its infectivity by soluble CD4 than parental virus. Furthermore, molecular dynamics simulations of the Env ectodomain (gp120 and gp41 ectodomain) bound with CD4 suggest that the three mutations increase binding affinity of Env for CD4 in solution. Thus, it is quite likely that the affinity for CD4 of the mutant Env is enhanced relative to the parental Env. Neutralization sensitivity of the triple mutant to CD4 binding site antibodies was not significantly different from that of parental virus, whereas the mutant exhibited a considerably higher resistance against neutralization by a CD4-induced epitope antibody and Env trimer-targeting V1/V2 antibodies. These results suggest that the three adaptive mutations cooperatively promote viral growth via increased CD4 affinity, and also that they enhance viral resistance to several neutralization antibodies by changing the Env-trimer conformation. In total, we have verified here an HIV-1 adaptation pathway in host cells and individuals involving Env derived from a lab-adapted and highly neutralization-sensitive clone.

In Vitro Neutralization Assay Using Cultured Hepatitis C Virus.

The method outlined here enables evaluation of the neutralization potency of monoclonal and polyclonal antibodies against in vitro cultured hepatitis C virus (HCV). The high variation in envelope protein sequence among HCV isolates necessitates the inclusion of several isolates, spanning the major genotypes of HCV, in order to make strong conclusions concerning the cross-reactive neutralization potential of a given antibody. This would be particularly relevant for any neutralization experiments aimed at uncovering novel therapeutic- or vaccine-relevant antibodies. In addition, these assays can also be used to compare neutralization sensitivity of novel cultured HCV to that of previously characterized isolates.

HIV-1 gp41 Residues Modulate CD4-Induced Conformational Changes in the Envelope Glycoprotein and Evolution of a Relaxed Conformation of gp120.

Entry of human immunodeficiency virus type 1 (HIV-1) into host cells is mediated by conformational changes in the envelope glycoprotein (Env) that are triggered by Env binding to cellular CD4 and chemokine receptors. These conformational changes involve the opening of the gp120 surface subunit, exposure of the fusion peptide in the gp41 transmembrane subunit, and refolding of the gp41 N- and C-terminal heptad repeat regions (HR1 and HR2) first into an extended prehairpin intermediate and then into a compact 6-helix bundle (6HB) that facilitates fusion between viral and host cell membranes. Previously, we reported that Envs resistant to HR1 peptide fusion inhibitors acquired key resistance mutations in either HR1 or HR2 that increased 6HB stability. Here, we identify residues in HR1 that contribute not only to fusion inhibitor resistance and 6HB stability but also to reduced reactivity to CD4-induced conformational changes that lead to 6HB formation. While all Envs show increased neutralization sensitivity to mimetic CD4 (mCD4), Envs with either the E560K or Q577R HR1 mutation reduced conformational reactivity to CD4 that resisted viral inactivation and triggering to the 6HB. Using a panel of monoclonal antibodies (mAbs), we further determined that Envs from both HR1 and HR2 resistance pathways exhibit a relaxed trimer conformation due to gp120 adaptive mutations in different regions of Env that segregate by resistance pathway. These findings highlight regions of cross talk between gp120 and gp41 and identify HR1 residues that play important roles in regulating CD4-induced conformational changes in Env.IMPORTANCE Binding of the HIV envelope glycoprotein (Env) to cellular CD4 and chemokine receptors triggers conformational changes in Env that mediate virus entry, but premature triggering of Env conformational changes leads to virus inactivation. Currently, we have a limited understanding of the network of residues that regulate Env conformational changes. Here, we identify residues in HR1 of gp41 that modulate conformational changes in response to gp120 binding to CD4 and show that the mutations in HR1 and HR2 that confer resistance to fusion inhibitors are associated with gp120 mutations in different regions of Env that confer a more open conformation. These findings contribute to our understanding of the regulation of Env conformational changes and efforts to design new entry inhibitors and stable Env vaccine immunogens.

Neutralization Sensitivity of a Novel HIV-1 CRF01_AE Panel of Infectious Molecular Clones.

HIV-1 CRF01_AE is dominant in Thailand where RV144 vaccine trial was conducted. To study immune correlates of protection in ongoing trials, CRF01_AE-derived reagents are essential. Here, we present a panel of 14 HIV-1 infectious molecular clones (IMCs) identified from different stages of infection and characterization of their neutralization sensitivity using 2 standard assays. One full-length IMC was constructed using a transmitted-founder virus to express Renilla luciferase (LucR) reporter gene and full-length envelopes (envs) of exogenous HIV-1. A panel of IMCs was generated, expressing envs of viruses from acute (Fiebig stages I/II and I-IV) and chronic (>Fiebig VI) infection. Neutralization assays were performed using TZM-bl or A3R5 cell lines, and sera or monoclonal antibodies (mAbs). Wilcoxon matched-paired test was used to assess neutralization differences between assays and reagents; correlation coefficients were evaluated by linear regression. Neutralization potency observed was significantly higher in the A3R5 assay when testing mAbs and serum pools (P < 0.0001); the stage of infection from which env was derived did not associate with IMC neutralization sensitivity. Neutralization values from A3R5 and TZM-bl assays were strongly correlated when mAbs were tested (R = 0.7, P < 0.0001), but a weaker association was seen with serum pools (R = 0.17, P = 0.03). This novel panel of CRF01_AE reporter IMC is useful for assessing vaccine-induced neutralizing antibodies in multiple assays, including those using primary cell targets. The significant differences in TZM-bl and A3R5 neutralization sensitivity, as well as the poor association when using polyclonal sera indicates the need for caution in choosing one specific platform.

Development and application of a monoclonal antibody-based blocking ELISA for detection of antibodies to Tembusu virus in multiple poultry species

BackgroundTembusu virus (TMUV) is a member of the genus Flavivirus. Outbreak of this virus infection in duck flocks was first observed in China in April 2010, causing severe egg drop and neurological signs in laying ducks. Recently reported duck infections in southeastern Asia highlighted the need for well-validated diagnostic methods of TMUV surveillance to understand its epidemiological characteristics and maintenance in nature. Several enzyme-linked immunosorbent assays (ELISAs) for the detection of TMUV infection have been reported, but none have been applied to high-throughput diagnostics.ResultsIn this study, a monoclonal antibody (MAb) against TMUV was generated and characterized. MAb 9E4 was shown to bind specifically to a disulfide bond-dependent epitope on the domain I/II of TMUV E protein, and a blocking ELISA was established based on this MAb. The cut-off percentage inhibition value for negative sera was set at 30%. By comparison with the virus neutralization test, the specificity and sensitivity of the blocking ELISA were 96.37% and 100%, respectively, and the kappa value was 0.966, based on 416 serum samples collected from both experimentally and clinically infected ducks, geese and chickens. A good correlation (r2 = 07998, P < 0.001) was observed between the blocking ELISA and plaque reduction neutralization test (PRNT) titers. Using archived duck serum samples collected between 2009 and 2015, the seroprevalence in duck flocks raised in Northern China was estimated by blocking ELISA.ConclusionsOur MAb-based blocking ELISA provides a reliable and rapid diagnostic tool for serological monitoring of TMUV infection and evaluation of immune status following TMUV vaccination in multiple poultry species.

Impact of Naturally Occurring Variation in the Human Papillomavirus 58 Capsid Proteins on Recognition by Type-Specific Neutralizing Antibodies.

Naturally occurring variants of human papillomavirus (HPV) 58 have been defined as lineages and sublineages but little is known about the impact of this diversity on protein function. We investigated the impact of variation within the major (L1) and minor (L2) capsid proteins of HPV58 on susceptibility to neutralizing antibodies. Pseudovirus (PsV) representing A1, A2, A3, B1, B2, C, D1, and D2 variants were evaluated for their susceptibility to antibodies elicited during natural infection, preclinical antisera generated against virus-like particles, and monoclonal antibodies (MAbs). Lineage C PsV demonstrated a decreased sensitivity to antibodies raised against lineage A antigens. Exchange of the DE, FG, and/or HI loops between sublineage A1 and lineage C demonstrated that residues within all 3 loops were essential for the differential sensitivity to natural infection antibodies, with slightly different requirements for the animal antisera and MAbs. Comparison between the HPV58 A1 L1 pentamer crystal structure and an HPV58 C homology model indicated that these differences in neutralization sensitivity were likely due to subtle epitope sequence changes rather that major structural alterations. These data improve our understanding of the impact of natural variation on HPV58 capsid antigenicity and raise the possibility of lineage-specific serotypes.

O‐001: Investigation of the molecular mechanisms that determine isolate‐specific differences in neutralization sensitivity of HCV

Journal of Viral HepatitisVolume 25, Issue S2 p. 6-6 Abstracts O-001: Investigation of the molecular mechanisms that determine isolate-specific differences in neutralization sensitivity of HCV First published: 12 June 2018 https://doi.org/10.1111/jvh.12922Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume25, IssueS2Special Issue: 16th International Symposium on Viral Hepatitis and Liver Diseases (ISVHLD) held from June 14th to 17th, 2018, in Toronto, CanadaJune 2018Pages 6-6 RelatedInformation

Conformational Stability of the Hemagglutinin of H5N1 Influenza A Viruses Influences Susceptibility to Broadly Neutralizing Stem Antibodies.

ABSTRACTVaccines that elicit broadly neutralizing antibodies to the conserved stem of hemagglutinin (HA) are being developed as universal influenza vaccines that protect against influenza across multiple years. However, different influenza virus strains, even those in the same subtype with identical stem sequences, can vary in susceptibility to broadly neutralizing stem antibodies, and the reasons are not understood. Here we studied potential mechanisms underlying the differing sensitivities of a panel of H5N1 HA pseudoviruses to broadly neutralizing stem antibodies. We found that greater HA conformational stability, as measured by thermal inactivation and pH triggering of conformational changes, correlates with reduced neutralization sensitivity and antibody binding to HA under neutral- and low-pH conditions. Our data indicate that the conformational stability of HA is an important attribute of susceptibility to broadly neutralizing stem antibodies and is influenced by residues outside the stem antibody epitopes.IMPORTANCE The influenza virus surface glycoprotein hemagglutinin (HA) mediates virus attachment and membrane fusion between virus and host cells, allowing the viral core to enter the host cell cytoplasm for replication. Fusion occurs when HA undergoes low-pH-induced-conformational changes during endocytosis. Broadly neutralizing antibodies targeted to the conserved stem region of HA interfere with conformational changes required for fusion. Vaccines that elicit such antibodies are being developed as novel universal influenza vaccines for multiyear protection. We investigated why H5N1 HAs from different strains differ in their sensitivity to broadly neutralizing stem antibodies despite having conserved epitopes. We report that HA conformational stability due to residues outside the antibody binding site accounted for much of the variation in susceptibility to neutralization by stem antibodies. These findings highlight the importance of nonepitope residues in influencing neutralization sensitivity to stem antibodies and the complexities in developing universal vaccines targeting conserved epitopes in the HA stem.

Comparison of Uncleaved and Mature Human Immunodeficiency Virus Membrane Envelope Glycoprotein Trimers.

The mature envelope glycoprotein (Env) spike on the surfaces of human immunodeficiency virus type 1 (HIV-1)-infected cells and virions is derived from proteolytic cleavage of a trimeric gp160 glycoprotein precursor. In these studies, we compared the conformations of cleaved and uncleaved membrane Envs with truncated cytoplasmic tails to those of stabilized soluble gp140 SOSIP.664 Env trimers. Deletion of the gp41 cytoplasmic tail did not significantly affect the sensitivity of viruses with the HIV-1AD8 Env to inhibition by antibodies or a CD4-mimetic compound. After glutaraldehyde fixation and purification from membranes, a cleaved Env exhibited a hydrodynamic radius of ∼10 nm and an antibody-binding profile largely consistent with that expected based on virus neutralization sensitivity. The purified cleaved Env trimers exhibited a hollow architecture with a central void near the trimer axis. Uncleaved Env, cross-linked and purified in parallel, exhibited a hydrodynamic radius similar to that of the cleaved Env. However, the uncleaved Env was recognized by poorly neutralizing antibodies and appeared by negative-stain electron microscopy to sample multiple conformations. Compared with membrane Envs, stabilized soluble gp140 SOSIP.664 Env trimers appear to be more compact, as reflected in their smaller hydrodynamic radii and negative-stain electron microscopy structures. The antigenic features of the soluble gp140 SOSIP.664 Env trimers differed from those of the cleaved membrane Env, particularly in gp120 V3 and some CD4-binding-site epitopes. Thus, proteolytic maturation allows the membrane-anchored Env to achieve a conformation that retains functional metastability but masks epitopes for poorly neutralizing antibodies.IMPORTANCE The entry of human immunodeficiency virus type 1 (HIV-1) into host cells is mediated by the envelope glycoprotein (Env) spike on the surface of the virus. Host antibodies elicited during natural HIV-1 infection or by vaccination can potentially recognize the Env spike and block HIV-1 infection. However, the changing shape of the HIV-1 Env spike protects the virus from antibody binding. Understanding the shapes of natural and man-made preparations of HIV-1 Envs will assist the development of effective vaccines against the virus. Here, we evaluate the effects of several Env modifications commonly used to produce Env preparations for vaccine studies and the determination of structure. We found that the cleavage of the HIV-1 Env precursor helps Env to assume its natural shape, which resists the binding of many commonly elicited antibodies. Stabilized soluble Envs exhibit more compact shapes but expose some Env elements differently than the natural Env.

Comparison of the genotypic and phenotypic properties of HIV-1 standard subtype B and subtype B/B′ env molecular clones derived from infections in China

Although a number of standardized human immunodeficiency virus 1 (HIV-1) pseudoviruses have been generated to assess neutralizing antibodies, subtype B/B′ has not been comprehensively characterized either genotypically or phenotypically. Full-length env genes were isolated from plasma samples derived from B/B′-infected individuals, including former plasma donors and men who had sex with men. The neutralization properties of these pseudoviruses were determined by testing against sCD4, 16 neutralizing monoclonal antibodies and 44 plasma samples, including samples from those infected with the three major prevalent clades in China. Twenty-eight pseudoviruses were successfully constructed, including 15 B′ and 13 B strains. Compared with subtype B strains from North America and Europe, the B′ strains from China showed greater variation in the V3 loop and higher susceptibility to the neutralizing antibody 2F5 and B/B′ plasma samples. The B′ strains from China showed significantly lower susceptibility to some trimer apex-binding neutralizing antibodies (PGT145, CH01, CH02, CH03, and CH04) than the B strains from Western countries. The 28 B-pseudotyped and B′-pseudotyped viruses were grouped into high, medium, and low clusters based on their overall neutralization sensitivity to plasma samples. The different genotypic and phenotypic properties of the standard subtype B from those of the Western viruses compared to the B and B′ strains from China suggest that clones from HIV-1-infected individuals in China are more suitable for the evaluation of candidate vaccines targeting the subtype B/B′ viruses circulating in China.

Infant transmitted/founder HIV-1 viruses from peripartum transmission are neutralization resistant to paired maternal plasma

Despite extensive genetic diversity of HIV-1 in chronic infection, a single or few maternal virus variants become the founders of an infant’s infection. These transmitted/founder (T/F) variants are of particular interest, as a maternal or infant HIV vaccine should raise envelope (Env) specific IgG responses capable of blocking this group of viruses. However, the maternal or infant factors that contribute to selection of infant T/F viruses are not well understood. In this study, we amplified HIV-1 env genes by single genome amplification from 16 mother-infant transmitting pairs from the U.S. pre-antiretroviral era Women Infant Transmission Study (WITS). Infant T/F and representative maternal non-transmitted Env variants from plasma were identified and used to generate pseudoviruses for paired maternal plasma neutralization sensitivity analysis. Eighteen out of 21 (85%) infant T/F Env pseudoviruses were neutralization resistant to paired maternal plasma. Yet, all infant T/F viruses were neutralization sensitive to a panel of HIV-1 broadly neutralizing antibodies and variably sensitive to heterologous plasma neutralizing antibodies. Also, these infant T/F pseudoviruses were overall more neutralization resistant to paired maternal plasma in comparison to pseudoviruses from maternal non-transmitted variants (p = 0.012). Altogether, our findings suggest that autologous neutralization of circulating viruses by maternal plasma antibodies select for neutralization-resistant viruses that initiate peripartum transmission, raising the speculation that enhancement of this response at the end of pregnancy could further reduce infant HIV-1 infection risk.

Distinct functions for the membrane-proximal ectodomain region (MPER) of HIV-1 gp41 in cell-free and cell–cell viral transmission and cell–cell fusion

HIV-1 is spread by cell-free virions and by cell-cell viral transfer. We asked whether the structure and function of a broad neutralizing antibody (bNAb) epitope, the membrane-proximal ectodomain region (MPER) of the viral gp41 transmembrane glycoprotein, differ in cell-free and cell-cell-transmitted viruses and whether this difference could be related to Ab neutralization sensitivity. Whereas cell-free viruses bearing W666A and I675A substitutions in the MPER lacked infectivity, cell-associated mutant viruses were able to initiate robust spreading infection. Infectivity was restored to cell-free viruses by additional substitutions in the cytoplasmic tail (CT) of gp41 known to disrupt interactions with the viral matrix protein. We observed contrasting effects on cell-free virus infectivity when W666A was introduced to two transmitted/founder isolates, but both mutants could still mediate cell-cell spread. Domain swapping indicated that the disparate W666A phenotypes of the cell-free transmitted/founder viruses are controlled by sequences in variable regions 1, 2, and 4 of gp120. The sequential passaging of an MPER mutant (W672A) in peripheral blood mononuclear cells enabled selection of viral revertants with loss-of-glycan suppressor mutations in variable region 1, suggesting a functional interaction between variable region 1 and the MPER. An MPER-directed bNAb neutralized cell-free virus but not cell-cell viral spread. Our results suggest that the MPER of cell-cell-transmitted virions has a malleable structure that tolerates mutagenic disruption but is not accessible to bNAbs. In cell-free virions, interactions mediated by the CT impose an alternative MPER structure that is less tolerant of mutagenic alteration and is efficiently targeted by bNAbs.

Identification of Novel Structural Determinants in MW965 Env That Regulate the Neutralization Phenotype and Conformational Masking Potential of Primary HIV-1 Isolates.

The subtype C HIV-1 isolate MW965.26 is a highly neutralization-sensitive tier 1a primary isolate that is widely used in vaccine studies, but the basis for the sensitive neutralization phenotype of this isolate is not known. Substituting the MW965.26 V1/V2 domain into a neutralization-sensitive SF162 Env clone resulted in high resistance to standard anti-V3 monoclonal antibodies, demonstrating that this region possesses strong masking activity in a standard Env backbone and indicating that determinants elsewhere in MW965.26 Env are responsible for its unusual neutralization sensitivity. Key determinants for this phenotype were mapped by generating chimeric Envs between MW965.26 Env and a typical resistant Env clone, the consensus C (ConC) clone, and localized to two residues, Cys384 in the C3 domain and Asn502 in the C5 domain. Substituting the sensitizing mutations Y384C and K502N at these positions into several resistant primary Envs resulted in conversion to neutralization-sensitive phenotypes, demonstrating the generalizability of this effect. In contrast to the sensitizing effects of these substitutions on normally masked epitopes, these mutations reduced the sensitivity of VRC01-like epitopes overlapping the CD4-binding domain, while they had no effect on several other classes of broadly neutralizing epitopes, including members of several lineages of V2-dependent quaternary epitopes and representatives of N332 glycan-dependent epitopes (PGT121) and quaternary, cleavage-dependent epitopes centered at the gp41-gp120 interface on intact HIV-1 Env trimers (PGT151). These results identify novel substitutions in gp120 that regulate the expression of alternative conformations of Env and differentially affect the exposure of different classes of epitopes, thereby influencing the neutralization phenotype of primary HIV-1 isolates.IMPORTANCE A better understanding of the mechanisms that determine the wide range of neutralization sensitivity of circulating primary HIV-1 isolates would provide important information about the natural structural and conformational diversity of HIV-1 Env and how this affects the neutralization phenotype. A useful way of studying this is to determine the molecular basis for the unusually high neutralization sensitivities of the limited number of available tier 1a viruses. This study localized the neutralization sensitivity of MW965.26, an extremely sensitive subtype C-derived primary isolate, to two rare substitutions in the C3 and C5 domains and demonstrated that the sequences at these positions differentially affect the presentation of epitopes recognized by different classes of standard and conformation-dependent broadly neutralizing antibodies. These results provide novel insight into how these regions regulate the neutralization phenotype and provide tools for controlling the Env conformation that could have applications both for structural studies and in vaccine design.

Alterations of HIV-1 envelope phenotype and antibody-mediated neutralization by signal peptide mutations.

HIV-1 envelope glycoprotein (Env) mediates virus attachment and entry into the host cells. Like other membrane-bound and secreted proteins, HIV-1 Env contains at its N terminus a signal peptide (SP) that directs the nascent Env to the endoplasmic reticulum (ER) where Env synthesis and post-translational modifications take place. SP is cleaved during Env biosynthesis but potentially influences the phenotypic traits of the Env protein. The Env SP sequences of HIV-1 isolates display high sequence variability, and the significance of such variability is unclear. We postulate that changes in the Env SP influence Env transport through the ER-Golgi secretory pathway and Env folding and/or glycosylation that impact on Env incorporation into virions, receptor binding and antibody recognition. We first evaluated the consequences of mutating the charged residues in the Env SP in the context of infectious molecular clone HIV-1 REJO.c/2864. Results show that three different mutations affecting histidine at position 12 affected Env incorporation into virions that correlated with reduction of virus infectivity and DC-SIGN-mediated virus capture and transmission. Mutations at positions 8, 12, and 15 also rendered the virus more resistant to neutralization by monoclonal antibodies against the Env V1V2 region. These mutations affected the oligosaccharide composition of N-glycans as shown by changes in Env reactivity with specific lectins and by mass spectrometry. Increased neutralization resistance and N-glycan composition changes were also observed when analogous mutations were introduced to another HIV-1 strain, JRFL. To the best of our knowledge, this is the first study showing that certain residues in the HIV-1 Env SP can affect virus neutralization sensitivity by modulating oligosaccharide moieties on the Env N-glycans. The HIV-1 Env SP sequences thus may be under selective pressure to balance virus infectiousness with virus resistance to the host antibody responses. (289 words)

Truncating the gp41 Cytoplasmic Tail of Simian Immunodeficiency Virus Decreases Sensitivity to Neutralizing Antibodies without Increasing the Envelope Content of Virions.

An incomplete understanding of native human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) envelope glycoproteins (Envs) impedes the development of structural models of Env and vaccine design. This shortcoming is due in part to the low number of Env trimers on virus particles. For SIV, this low expression level can be counteracted by truncating the cytoplasmic tail (CT) of Env. CT truncation has been shown to increase Env incorporation into the virion and is commonly used in vaccine and imaging studies, but its effects on viral antigenicity have not been fully elucidated. To study the effects of a CT truncation of Env in viruses in similar genetic contexts, we introduced stop codons into the CT of a SIVsmE660 molecular clone and two neutralizing antibody (NAb) escape variants. These viruses shared 98% sequence identity in Env but were characterized as either tier 1 (sensitive to neutralization), tier 2 (moderately resistant to neutralization), or tier 3 (resistant to neutralization). However, the introduction of premature stop codons in Env at position Q741/Q742 converted all three transfection-derived viruses to a tier 3-like phenotype, and these viruses were uniformly resistant to neutralization by sera from infected macaques and monoclonal antibodies (MAbs). These changes in neutralization sensitivity were not accompanied by an increase in either the virion Env content of infection-derived viruses or the infectivity of transfection-derived viruses in human cells, suggesting that CT mutations may result in global changes to the Env conformation. Our results demonstrate that some CT truncations can affect viral antigenicity and, as such, may not be suitable surrogate models of native HIV/SIV Env.IMPORTANCE Modifications to the SIV envelope protein (Env) are commonly used in structural and vaccine studies to stabilize and increase the expression of Env, often without consideration of effects on antigenicity. One such widespread modification is the truncation of the Env C-terminal tail. Here, we studied the effects of a particular cytoplasmic tail truncation in three SIVsm strains that have highly similar Env sequences but exhibit different sensitivities to neutralizing antibodies. After truncation of the Env CT, these viruses were all very resistant to neutralization by sera from infected macaques and monoclonal antibodies. The viruses with a truncated Env CT also did not exhibit the desired and typical increase in Env expression. These results underscore the importance of carefully evaluating the use of truncated Env as a model in HIV/SIV vaccine and imaging studies and of the continued need to find better models of native Env that contain fewer modifications.

The Zika virus envelope protein glycan loop regulates virion antigenicity

Because antibodies are an important component of flavivirus immunity, understanding the antigenic structure of flaviviruses is critical. Compared to dengue virus (DENV), the loop containing the single N-linked glycosylation site on Zika virus (ZIKV) envelope (E) proteins extends further towards the DII fusion loop (DII-FL) on neighboring E proteins within E dimers on mature viruses. Although ZIKV is poorly neutralized by DII-FL antibodies, we demonstrated significantly increased neutralization sensitivity of ZIKV particles incorporating the DENV glycan loop. Increased neutralization sensitivity was independent of E protein glycosylation: ZIKV lacking E protein glycans remained poorly neutralized, whereas ZIKV loop chimeras with or without an E protein glycan were potently neutralized. ZIKV particles lacking the E protein glycan were capable of infecting Raji cells expressing the lectin DC-SIGNR, suggesting the prM glycan of partially mature particles can facilitate entry. Our study provides insight into the determinants of ZIKV E protein function and antigenicity.