Potent neutralizing antibodies elicited by dengue vaccine in rhesus macaque target diverse epitopes.

There is still no safe and effective vaccine against dengue virus infection. Epidemics of dengue virus infection are increasingly a threat to human health around the world. Antibodies generated in response to dengue infection have been shown to impact disease development and effectiveness of dengue vaccine. In this study, we investigated monoclonal antibody responses to an experimental dengue vaccine in rhesus macaques. Variable regions of both heavy chain (VH) and light chain (VL) were cloned from single antibody-secreting B cells. A total of 780 monoclonal antibodies (mAbs) composed of paired VH and VL were characterized. Results show that the vaccination induces mAbs with diverse germline sequences and a wide range of binding affinities. Six potent neutralizing mAbs were identified among 130 dengue envelope protein binders. Critical amino acids for each neutralizing antibody binding to the dengue envelope protein were identified by alanine scanning of mutant libraries. Diverse epitopes were identified, including epitopes on the lateral ridge of DIII, the I-III hinge, the bc loop adjacent to the fusion loop of DII, and the ß-strands and loops of DI. Significantly, one of the neutralizing mAbs has a previously unknown epitope in DII at the interface of the envelope and membrane protein and is capable of neutralizing all four dengue serotypes. Taken together, the results of this study not only provide preclinical validation for the tested experimental vaccine, but also shed light on a potential application of the rhesus macaque model for better dengue vaccine evaluation and design of vaccines and immunization strategies.

SELfies and CELLfies: Whole Genome Sequencing and Annotation of Five Antibiotic Resistant Bacteria Isolated from the Surfaces of Smartphones, An Inquiry Based Laboratory Exercise in a Genomics Undergraduate Course at the Rochester Institute of Technology.

Are touchscreen devices a public health risk for the transmission of pathogenic bacteria, especially those that are resistant to antibiotics? To investigate this, we embarked on a project aimed at isolating and identifying bacteria that are resistant to antibiotics from the screens of smartphones. Touchscreen devices have become ubiquitous in society, and it is important to evaluate the potential risks they pose towards public health, especially as it pertains to the harboring and transmission of pathogenic bacteria that are resistant to antibiotics. Sixteen bacteria were initially isolated of which five were unique (four Staphylococcus species and one Micrococcus species). The genomes of the five unique isolates were subsequently sequenced and annotated. The genomes were analyzed using in silico tools to predict the synthesis of antibiotics and secondary metabolites using the antibiotics and Secondary Metabolite Analysis SHell (antiSMASH) tool in addition to the presence of gene clusters that denote resistance to antibiotics using the Resistance Gene Identifier (RGI) tool. In vivo analysis was also done to assess resistance/susceptibility to four antibiotics that are commonly used in a research laboratory setting. The data presented in this manuscript is the result of a semester-long inquiry based laboratory exercise in the genomics course (BIOL340) in the Thomas H. Gosnell School of Life Sciences/College of Science at the Rochester Institute of Technology.

Probing the antigenicity of hepatitis C virus envelope glycoprotein complex by high-throughput mutagenesis.

The hepatitis C virus (HCV) envelope glycoproteins E1 and E2 form a non-covalently linked heterodimer on the viral surface that mediates viral entry. E1, E2 and the heterodimer complex E1E2 are candidate vaccine antigens, but are technically challenging to study because of difficulties in producing natively folded proteins by standard protein expression and purification methods. To better comprehend the antigenicity of these proteins, a library of alanine scanning mutants comprising the entirety of E1E2 (555 residues) was created for evaluating the role of each residue in the glycoproteins. The mutant library was probed, by a high-throughput flow cytometry-based assay, for binding with the co-receptor CD81, and a panel of 13 human and mouse monoclonal antibodies (mAbs) that target continuous and discontinuous epitopes of E1, E2, and the E1E2 complex. Together with the recently determined crystal structure of E2 core domain (E2c), we found that several residues in the E2 back layer region indirectly impact binding of CD81 and mAbs that target the conserved neutralizing face of E2. These findings highlight an unexpected role for the E2 back layer in interacting with the E2 front layer for its biological function. We also identified regions of E1 and E2 that likely located at or near the interface of the E1E2 complex, and determined that the E2 back layer also plays an important role in E1E2 complex formation. The conformation-dependent reactivity of CD81 and the antibody panel to the E1E2 mutant library provides a global view of the influence of each amino acid (aa) on E1E2 expression and folding. This information is valuable for guiding protein engineering efforts to enhance the antigenic properties and stability of E1E2 for vaccine antigen development and structural studies.

Broadly neutralizing antibodies with few somatic mutations and hepatitis C virus clearance.

Here, we report the isolation of broadly neutralizing mAbs (bNAbs) from persons with broadly neutralizing serum who spontaneously cleared hepatitis C virus (HCV) infection. We found that bNAbs from two donors bound the same epitope and were encoded by the same germline heavy chain variable gene segment. Remarkably, these bNAbs were encoded by antibody variable genes with sparse somatic mutations. For one of the most potent bNAbs, these somatic mutations were critical for antibody neutralizing breadth and for binding to autologous envelope variants circulating late in infection. However, somatic mutations were not necessary for binding of the bNAb unmutated ancestor to envelope proteins of early autologous transmitted/founder viruses. This study identifies a public B cell clonotype favoring early recognition of a conserved HCV epitope, proving that anti-HCV bNAbs can achieve substantial neutralizing breadth with relatively few somatic mutations, and identifies HCV envelope variants that favored selection and maturation of an anti-HCV bNAb in vivo. These data provide insight into the molecular mechanisms of immune-mediated clearance of HCV infection and present a roadmap to guide development of a vaccine capable of stimulating anti-HCV bNAbs with a physiologic number of somatic mutations characteristic of vaccine responses.

Mapping the Human Memory B Cell and Serum Neutralizing Antibody Responses to Dengue Virus Serotype 4 Infection and Vaccination.

The four dengue virus (DENV) serotypes are mosquito-borne flaviviruses responsible for dengue fever and dengue hemorrhagic fever. People exposed to DENV develop antibodies (Abs) that strongly neutralize the serotype responsible for infection. Historically, infection with DENV serotype 4 (DENV4) has been less common and less studied than infections with the other three serotypes. However, DENV4 has been responsible for recent large and sustained epidemics in Asia and Latin America. The neutralizing antibody responses and the epitopes targeted against DENV4 have not been characterized in human infection. In this study, we mapped and characterized epitopes on DENV4 recognized by neutralizing antibodies in people previously exposed to DENV4 infections or to a live attenuated DENV4 vaccine. To study the fine specificity of DENV4 neutralizing human antibodies, B cells from two people exposed to DENV4 were immortalized and screened to identify DENV-specific clones. Two human monoclonal antibodies (MAbs) that neutralized DENV4 were isolated, and their epitopes were finely mapped using recombinant viruses and alanine scan mutation array techniques. Both antibodies bound to quaternary structure epitopes near the hinge region between envelope protein domain I (EDI) and EDII. In parallel, to characterize the serum neutralizing antibody responses, convalescence-phase serum samples from people previously exposed to primary DENV4 natural infections or a monovalent DENV4 vaccine were analyzed. Natural infection and vaccination also induced serum-neutralizing antibodies that targeted similar epitope domains at the EDI/II hinge region. These studies defined a target of neutralizing antigenic site on DENV4 targeted by human antibodies following natural infection or vaccination.IMPORTANCE The four serotypes of dengue virus are the causative agents of dengue fever and dengue hemorrhagic fever. People exposed to primary DENV infections develop long-term neutralizing antibody responses, but these principally recognize only the infecting serotype. An effective vaccine against dengue should elicit long-lasting protective antibody responses to all four serotypes simultaneously. We and others have defined antigenic sites on the envelope (E) protein of viruses of dengue virus serotypes 1, 2, and 3 targeted by human neutralizing antibodies. The epitopes on DENV4 E protein targeted by the human neutralizing antibodies and the mechanisms of serotype 4 neutralization are poorly understood. Here, we report the properties of human antibodies that neutralize dengue virus serotype 4. People exposed to serotype 4 infections or a live attenuated serotype 4 vaccine developed neutralizing antibodies that bound to similar sites on the viral E protein. These studies have provided a foundation for developing and evaluating DENV4 vaccines.

Protective Capacity of the Human Anamnestic Antibody Response during Acute Dengue Virus Infection.

Half of the world's population is exposed to the risk of dengue virus infection. Although a vaccine for dengue virus is now available in a few countries, its reported overall efficacy of about 60% is not ideal. Protective immune correlates following natural dengue virus infection remain undefined, which makes it difficult to predict the efficacy of new vaccines. In this study, we address the protective capacity of dengue virus-specific antibodies that are produced by plasmablasts a few days after natural secondary infection. Among a panel of 18 dengue virus-reactive human monoclonal antibodies, four groups of antibodies were identified based on their binding properties. While antibodies targeting the fusion loop of the glycoprotein of dengue virus dominated the antibody response, two smaller groups of antibodies bound to previously undescribed epitopes in domain II of the E protein. The latter, largely serotype-cross-reactive antibodies, demonstrated increased stability of binding at pH 5. These antibodies possessed weak to moderate neutralization capacity in vitro but were the most efficacious in promoting the survival of infected mice. Our data suggest that the cross-reactive anamnestic antibody response has a protective capacity despite moderate neutralization in vitro and a moderate decrease of viremia in vivo IMPORTANCE: Antibodies can protect from symptomatic dengue virus infection. However, it is not easy to assess which classes of antibodies provide protection because in vitro assays are not always predictive of in vivo protection. During a repeat infection, dengue virus-specific immune memory cells are reactivated and large amounts of antibodies are produced. By studying antibodies cloned from patients with heterologous secondary infection, we tested the protective value of the serotype-cross-reactive "recall" or "anamnestic" response. We found that results from in vitro neutralization assays did not always correlate with the ability of the antibodies to reduce viremia in a mouse model. In addition, a decrease of viremia in mice did not necessarily improve survival. The most protective antibodies were stable at pH 5, suggesting that antibody binding in the endosomes, after the antibody-virus complex is internalized, might be important to block virus spread in the organism.

Functional Transplant of a Dengue Virus Serotype 3 (DENV3)-Specific Human Monoclonal Antibody Epitope into DENV1.

UNLABELLED: The four dengue virus (DENV) serotypes, DENV1 through 4, are endemic throughout tropical and subtropical regions of the world. While first infection confers long-term protective immunity against viruses of the infecting serotype, a second infection with virus of a different serotype carries a greater risk of severe dengue disease, including dengue hemorrhagic fever and dengue shock syndrome. Recent studies demonstrate that humans exposed to DENV infections develop neutralizing antibodies that bind to quaternary epitopes formed by the viral envelope (E) protein dimers or higher-order assemblies required for the formation of the icosahedral viral envelope. Here we show that the quaternary epitope target of the human DENV3-specific neutralizing monoclonal antibody (MAb) 5J7 can be partially transplanted into a DENV1 strain by changing the core residues of the epitope contained within a single monomeric E molecule. MAb 5J7 neutralized the recombinant DENV1/3 strain in cell culture and was protective in a mouse model of infection with the DENV1/3 strain. However, the 5J7 epitope was only partially recreated by transplantation of the core residues because MAb 5J7 bound and neutralized wild-type (WT) DENV3 better than the DENV1/3 recombinant. Our studies demonstrate that it is possible to transplant a large number of discontinuous residues between DENV serotypes and partially recreate a complex antibody epitope, while retaining virus viability. Further refinement of this approach may lead to new tools for measuring epitope-specific antibody responses and new vaccine platforms. IMPORTANCE: Dengue virus is the most important mosquito-borne pathogen of humans worldwide, with approximately one-half the world's population living in regions where dengue is endemic. Dengue immunity following infection is robust and thought to be conferred by antibodies raised against the infecting virus. However, the specific viral components that these antibodies recognize and how they neutralize the virus have been incompletely described. Here we map a region on dengue virus serotype 3 recognized by the human neutralizing antibody 5J7 and then test the functional significance of this region by transplanting it into a serotype 1 virus. Our studies demonstrate a region on dengue virus necessary for 5J7 binding and neutralization. Our work also demonstrates the technical feasibility of engineering dengue viruses to display targets of protective antibodies. This technology can be used to develop new dengue vaccines and diagnostic assays.

Lower IgG somatic hypermutation rates during acute dengue virus infection is compatible with a germinal center-independent B cell response.

BACKGROUND: The study of human B cell response to dengue virus (DENV) infection is critical to understand serotype-specific protection and the cross-reactive sub-neutralizing response. Whereas the first is beneficial and thus represents the ultimate goal of vaccination, the latter has been implicated in the development of severe disease, which occurs in a small, albeit significant, fraction of secondary DENV infections. Both primary and secondary infections are associated with the production of poly-reactive and cross-reactive IgG antibodies. METHODS: To gain insight into the effect of DENV infection on the B cell repertoire, we used VH region high-throughput cDNA sequencing of the peripheral blood IgG B cell compartment of 19 individuals during the acute phase of infection. For 11 individuals, a second sample obtained 6 months later was analyzed for comparison. Probabilities of sequencing antibody secreting cells or memory B cells were estimated using second-order Monte Carlo simulation. RESULTS: We found that in acute disease there is an increase in IgG B cell diversity and changes in the relative use of segments IGHV1-2, IGHV1-18, and IGHV1-69. Somewhat unexpectedly, an overall low proportion of somatic hypermutated antibody genes was observed during the acute phase plasmablasts, particularly in secondary infections and those cases with more severe disease. CONCLUSIONS: Our data are consistent with an innate-like antiviral recognition system mediated by B cells using defined germ-line coded B cell receptors, which could provide a rapid germinal center-independent antibody response during the early phase of infection. A model describing concurrent T-dependent and T-independent B cell responses in the context of DENV infection is proposed, which incorporates the selection of B cells using hypomutated IGHV segments and their potential role in poly/cross-reactivity. Its formal demonstration could lead to a definition of its potential implication in antibody-dependent enhancement, and may contribute to rational vaccine development efforts.

Dengue Virus prM-Specific Human Monoclonal Antibodies with Virus Replication-Enhancing Properties Recognize a Single Immunodominant Antigenic Site.

UNLABELLED: The proposed antibody-dependent enhancement (ADE) mechanism for severe dengue virus (DENV) disease suggests that non-neutralizing serotype cross-reactive antibodies generated during a primary infection facilitate entry into Fc receptor bearing cells during secondary infection, resulting in enhanced viral replication and severe disease. One group of cross-reactive antibodies that contributes considerably to this serum profile target the premembrane (prM) protein. We report here the isolation of a large panel of naturally occurring human monoclonal antibodies (MAbs) obtained from subjects following primary DENV serotype 1, 2, or 3 or secondary natural DENV infections or following primary DENV serotype 1 live attenuated virus vaccination to determine the antigenic landscape on the prM protein that is recognized by human antibodies. We isolated 25 prM-reactive human MAbs, encoded by diverse antibody-variable genes. Competition-binding studies revealed that all of the antibodies bound to a single major antigenic site on prM. Alanine scanning-based shotgun mutagenesis epitope mapping studies revealed diverse patterns of fine specificity of various clones, suggesting that different antibodies use varied binding poses to recognize several overlapping epitopes within the immunodominant site. Several of the antibodies interacted with epitopes on both prM and E protein residues. Despite the diverse genetic origins of the antibodies and differences in the fine specificity of their epitopes, each of these prM-reactive antibodies was capable of enhancing the DENV infection of Fc receptor-bearing cells. IMPORTANCE: Antibodies may play a critical role in the pathogenesis of enhanced DENV infection and disease during secondary infections. A substantial proportion of enhancing antibodies generated in response to natural dengue infection are directed toward the prM protein. The fine specificity of human prM antibodies is not understood. Here, we isolated a panel of dengue prM-specific human monoclonal antibodies from individuals after infection in order to define the mode of molecular recognition by enhancing antibodies. We found that only a single antibody molecule can be bound to each prM protein at any given time. Distinct overlapping epitopes were mapped, but all of the epitopes lie within a single major antigenic site, suggesting that this antigenic domain forms an immunodominant region of the protein. Neutralization and antibody-dependent enhanced replication experiments showed that recognition of any of the epitopes within the major antigenic site on prM was sufficient to cause enhanced infection of target cells.

Mechanism of Binding to Ebola Virus Glycoprotein by the ZMapp, ZMAb, and MB-003 Cocktail Antibodies.

UNLABELLED: Cocktails of monoclonal antibodies (MAbs) that target the surface glycoprotein (GP) of Ebola virus (EBOV) are effective in nonhuman primate models and have been used under emergency compassionate-treatment protocols in human patients. However, the amino acids that form the detailed binding epitopes for the MAbs in the ZMapp, ZMAb, and the related MB-003 cocktails have yet to be identified. Other binding properties that define how each MAb functionally interacts with GP­such as affinity, epitope conservation, and epitope accessibility­also remain largely unknown. To help define how each MAb interacts with GP, here we used comprehensive alanine-scanning mutagenesis (shotgun mutagenesis), neutralization escape, and whole virion binding to define each MAb's specific epitope, epitope accessibility, epitope conservation, and apparent affinity. Each of the six therapeutic MAbs binds nonidentical epitopes in the GP base, glycan cap, or mucin-like domain. Their apparent affinity, epitope complementarity, and epitope accessibility helps explain why MAbs 4G7 and 13C6 are more protective than 2G4 and 1H3. The mucin-like domain MAbs 6D8 and 13F6 bind with the strongest apparent affinity, helping to explain their effectiveness in vivo despite their inability to neutralize virus. IMPORTANCE: Ebola virus disease (EVD) can be caused by four different filovirus family members, including Ebola virus (EBOV), which infected 10 times more people in western Africa over the last year than all previous EVD outbreaks combined, with a number of cases distributed across the globe by travelers. Cocktails of inhibitory monoclonal antibodies (MAbs), such as ZMAb, MB-003, and in particular ZMapp, have demonstrated in animal models some of the most significant therapeutic potential for treating EVD, and in 2014, 15 patients were treated with ZMapp or ZMAb under compassionate-use protocols. Here, we have defined the epitope features for the most important therapeutic MAbs against EBOV developed to date. Defining the epitopes and binding characteristics for these MAbs, as well as the commonly used reference MAb KZ52, helps explain their breadth of reactivity against different ebolavirus species, predict viral evasion against these MAbs, and design new cocktails of MAbs with improved complementarity.

Dengue virus envelope protein domain I/II hinge determines long-lived serotype-specific dengue immunity.

The four dengue virus (DENV) serotypes, DENV-1, -2, -3, and -4, are endemic throughout tropical and subtropical regions of the world, with an estimated 390 million acute infections annually. Infection confers long-term protective immunity against the infecting serotype, but secondary infection with a different serotype carries a greater risk of potentially fatal severe dengue disease, including dengue hemorrhagic fever and dengue shock syndrome. The single most effective measure to control this threat to global health is a tetravalent DENV vaccine. To date, attempts to develop a protective vaccine have progressed slowly, partly because the targets of type-specific human neutralizing antibodies (NAbs), which are critical for long-term protection, remain poorly defined, impeding our understanding of natural immunity and hindering effective vaccine development. Here, we show that the envelope glycoprotein domain I/II hinge of DENV-3 and DENV-4 is the primary target of the long-term type-specific NAb response in humans. Transplantation of a DENV-4 hinge into a recombinant DENV-3 virus showed that the hinge determines the serotype-specific neutralizing potency of primary human and nonhuman primate DENV immune sera and that the hinge region both induces NAbs and is targeted by protective NAbs in rhesus macaques. These results suggest that the success of live dengue vaccines may depend on their ability to stimulate NAbs that target the envelope glycoprotein domain I/II hinge region. More broadly, this study shows that complex conformational antibody epitopes can be transplanted between live viruses, opening up similar possibilities for improving the breadth and specificity of vaccines for influenza, HIV, hepatitis C virus, and other clinically important viral pathogens.

The potent and broadly neutralizing human dengue virus-specific monoclonal antibody 1C19 reveals a unique cross-reactive epitope on the bc loop of domain II of the envelope protein.

UNLABELLED: Following natural dengue virus (DENV) infection, humans produce some antibodies that recognize only the serotype of infection (type specific) and others that cross-react with all four serotypes (cross-reactive). Recent studies with human antibodies indicate that type-specific antibodies at high concentrations are often strongly neutralizing in vitro and protective in animal models. In general, cross-reactive antibodies are poorly neutralizing and can enhance the ability of DENV to infect Fc receptor-bearing cells under some conditions. Type-specific antibodies at low concentrations also may enhance infection. There is an urgent need to determine whether there are conserved antigenic sites that can be recognized by cross-reactive potently neutralizing antibodies. Here, we describe the isolation of a large panel of naturally occurring human monoclonal antibodies (MAbs) directed to the DENV domain II fusion loop (FL) envelope protein region from subjects following vaccination or natural infection. Most of the FL-specific antibodies exhibited a conventional phenotype, characterized by low-potency neutralizing function and antibody-dependent enhancing activity. One clone, however, recognized the bc loop of domain II adjacent to the FL and exhibited a unique phenotype of ultrahigh potency, neutralizing all four serotypes better than any other previously described MAb recognizing this region. This antibody not only neutralized DENV effectively but also competed for binding against the more prevalent poor-quality antibodies whose binding was focused on the FL. The 1C19 human antibody could be a promising component of a preventative or therapeutic intervention. Furthermore, the unique epitope revealed by 1C19 suggests a focus for rational vaccine design based on novel immunogens presenting cross-reactive neutralizing determinants. IMPORTANCE: With no effective vaccine available, the incidence of dengue virus (DENV) infections worldwide continues to rise, with more than 390 million infections estimated to occur each year. Due to the unique roles that antibodies are postulated to play in the pathogenesis of DENV infection and disease, there is consensus that a successful DENV vaccine must protect against all four serotypes. If conserved epitopes recognized by naturally occurring potently cross-neutralizing human antibodies could be identified, monovalent subunit vaccine preparations might be developed. We characterized 30 DENV cross-neutralizing human monoclonal antibodies (MAbs) and identified one (1C19) that recognized a novel conserved site, known as the bc loop. This antibody has several desirable features, as it neutralizes DENV effectively and competes for binding against the more common low-potency fusion loop (FL) antibodies, which are believed to contribute to antibody-mediated disease. To our knowledge, this is the first description of a potent serotype cross-neutralizing human antibody to DENV.

Atomic-level functional model of dengue virus Envelope protein infectivity.

A number of structures have been solved for the Envelope (E) protein from dengue virus and closely related flaviviruses, providing detailed pictures of the conformational states of the protein at different stages of infectivity. However, the key functional residues responsible for mediating the dynamic changes between these structures remain largely unknown. Using a comprehensive library of functional point mutations covering all 390 residues of the dengue virus E protein ectodomain, we identified residues that are critical for virus infectivity, but that do not affect E protein expression, folding, virion assembly, or budding. The locations and atomic interactions of these critical residues within different structures representing distinct fusogenic conformations help to explain how E protein (i) regulates fusion-loop exposure by shielding, tethering, and triggering its release; (ii) enables hinge movements between E domain interfaces during triggered structural transformations; and (iii) drives membrane fusion through late-stage zipper contacts with stem. These results provide structural targets for drug and vaccine development and integrate the findings from structural studies and isolated mutagenesis efforts into a cohesive model that explains how specific residues in this class II viral fusion protein enable virus infectivity.

Transmitted/founder and chronic subtype C HIV-1 use CD4 and CCR5 receptors with equal efficiency and are not inhibited by blocking the integrin α4ß7.

Sexual transmission of human immunodeficiency virus type 1 (HIV-1) most often results from productive infection by a single transmitted/founder (T/F) virus, indicating a stringent mucosal bottleneck. Understanding the viral traits that overcome this bottleneck could have important implications for HIV-1 vaccine design and other prevention strategies. Most T/F viruses use CCR5 to infect target cells and some encode envelope glycoproteins (Envs) that contain fewer potential N-linked glycosylation sites and shorter V1/V2 variable loops than Envs from chronic viruses. Moreover, it has been reported that the gp120 subunits of certain transmitted Envs bind to the gut-homing integrin α4ß7, possibly enhancing virus entry and cell-to-cell spread. Here we sought to determine whether subtype C T/F viruses, which are responsible for the majority of new HIV-1 infections worldwide, share biological properties that increase their transmission fitness, including preferential α4ß7 engagement. Using single genome amplification, we generated panels of both T/F (n = 20) and chronic (n = 20) Env constructs as well as full-length T/F (n = 6) and chronic (n = 4) infectious molecular clones (IMCs). We found that T/F and chronic control Envs were indistinguishable in the efficiency with which they used CD4 and CCR5. Both groups of Envs also exhibited the same CD4+ T cell subset tropism and showed similar sensitivity to neutralization by CD4 binding site (CD4bs) antibodies. Finally, saturating concentrations of anti-α4ß7 antibodies failed to inhibit infection and replication of T/F as well as chronic control viruses, although the growth of the tissue culture-adapted strain SF162 was modestly impaired. These results indicate that the population bottleneck associated with mucosal HIV-1 acquisition is not due to the selection of T/F viruses that use α4ß7, CD4 or CCR5 more efficiently.

Primary infection by a human immunodeficiency virus with atypical coreceptor tropism.

The great majority of human immunodeficiency virus type 1 (HIV-1) strains enter CD4+ target cells by interacting with one of two coreceptors, CCR5 or CXCR4. Here we describe a transmitted/founder (T/F) virus (ZP6248) that was profoundly impaired in its ability to utilize CCR5 and CXCR4 coreceptors on multiple CD4+ cell lines as well as primary human CD4+ T cells and macrophages in vitro yet replicated to very high titers (>80 million RNA copies/ml) in an acutely infected individual. Interestingly, the envelope (Env) glycoprotein of this clade B virus had a rare GPEK sequence in the crown of its third variable loop (V3) rather than the consensus GPGR sequence. Extensive sequencing of sequential plasma samples showed that the GPEK sequence was present in virtually all Envs, including those from the earliest time points after infection. The molecularly cloned (single) T/F virus was able to replicate, albeit poorly, in cells obtained from ccr5Δ32 homozygous donors. The ZP6248 T/F virus could also infect cell lines overexpressing the alternative coreceptors GPR15, APJ, and FPRL-1. A single mutation in the V3 crown sequence (GPEK->GPGK) of ZP6248 restored its infectivity in CCR5+ cells but reduced its ability to replicate in GPR15+ cells, indicating that the V3 crown motif played an important role in usage of this alternative coreceptor. These results suggest that the ZP6248 T/F virus established an acute in vivo infection by using coreceptor(s) other than CCR5 or CXCR4 or that the CCR5 coreceptor existed in an unusual conformation in this individual.

Phenotypic and immunologic comparison of clade B transmitted/founder and chronic HIV-1 envelope glycoproteins.

Sexual transmission of human immunodeficiency virus type 1 (HIV-1) across mucosal barriers is responsible for the vast majority of new infections. This relatively inefficient process results in the transmission of a single transmitted/founder (T/F) virus, from a diverse viral swarm in the donor, in approximately 80% of cases. Here we compared the biological activities of 24 clade B T/F envelopes (Envs) with those from 17 chronic controls to determine whether the genetic bottleneck that occurs during transmission is linked to a particular Env phenotype. To maximize the likelihood of an intact mucosal barrier in the recipients and to enhance the sensitivity of detecting phenotypic differences, only T/F Envs from individuals infected with a single T/F variant were selected. Using pseudotyping to assess Env function in single-round infectivity assays, we compared coreceptor tropism, CCR5 utilization efficiencies, primary CD4(+) T cell subset tropism, dendritic cell trans-infections, fusion kinetics, and neutralization sensitivities. T/F and chronic Envs were phenotypically equivalent in most assays; however, T/F Envs were modestly more sensitive to CD4 binding site antibodies b12 and VRC01, as well as pooled human HIV Ig. This finding was independently validated with a panel of 14 additional chronic HIV-1 Env controls. Moreover, the enhanced neutralization sensitivity was associated with more efficient binding of b12 and VRC01 to T/F Env trimers. These data suggest that there are subtle but significant structural differences between T/F and chronic clade B Envs that may have implications for HIV-1 transmission and the design of effective vaccines.

HIV-1 resistance to CCR5 antagonists associated with highly efficient use of CCR5 and altered tropism on primary CD4+ T cells.

We previously reported on a panel of HIV-1 clade B envelope (Env) proteins isolated from a patient treated with the CCR5 antagonist aplaviroc (APL) that were drug resistant. These Envs used the APL-bound conformation of CCR5, were cross resistant to other small-molecule CCR5 antagonists, and were isolated from the patient's pretreatment viral quasispecies as well as after therapy. We analyzed viral and host determinants of resistance and their effects on viral tropism on primary CD4(+) T cells. The V3 loop contained residues essential for viral resistance to APL, while additional mutations in gp120 and gp41 modulated the magnitude of drug resistance. However, these mutations were context dependent, being unable to confer resistance when introduced into a heterologous virus. The resistant virus displayed altered binding between gp120 and CCR5 such that the virus became critically dependent on the N' terminus of CCR5 in the presence of APL. In addition, the drug-resistant Envs studied here utilized CCR5 very efficiently: robust virus infection occurred even when very low levels of CCR5 were expressed. However, recognition of drug-bound CCR5 was less efficient, resulting in a tropism shift toward effector memory cells upon infection of primary CD4(+) T cells in the presence of APL, with relative sparing of the central memory CD4(+) T cell subset. If such a tropism shift proves to be a common feature of CCR5-antagonist-resistant viruses, then continued use of CCR5 antagonists even in the face of virologic failure could provide a relative degree of protection to the T(CM) subset of CD4(+) T cells and result in improved T cell homeostasis and immune function.