A Recombinant Chimeric Ad5/3 Vector Expressing a Multistage Plasmodium Antigen Induces Protective Immunity in Mice Using Heterologous Prime-Boost Immunization Regimens.

An ideal malaria vaccine should target several stages of the parasite life cycle and induce antiparasite and antidisease immunity. We have reported a Plasmodium yoelii chimeric multistage recombinant protein (P. yoelii linear peptide chimera/recombinant modular chimera), engineered to express several autologous T cell epitopes and sequences derived from the circumsporozoite protein and the merozoite surface protein 1. This chimeric protein elicits protective immunity, mediated by CD4(+) T cells and neutralizing Abs. However, experimental evidence, from pre-erythrocytic vaccine candidates and irradiated sporozoites, has shown that CD8(+) T cells play a significant role in protection. Recombinant viral vectors have been used as a vaccine platform to elicit effective CD8(+) T cell responses. The human adenovirus (Ad) serotype 5 has been tested in malaria vaccine clinical trials with excellent safety profile. Nevertheless, a major concern for the use of Ad5 is the high prevalence of anti-vector neutralizing Abs in humans, hampering its immunogenicity. To minimize the impact of anti-vector pre-existing immunity, we developed a chimeric Ad5/3 vector in which the knob region of Ad5 was replaced with that of Ad3, conferring partial resistance to anti-Ad5 neutralizing Abs. Furthermore, we implemented heterologous Ad/protein immunization regimens that include a single immunization with recombinant Ad vectors. Our data show that immunization with the recombinant Ad5/3 vector induces protective efficacy indistinguishable from that elicited by Ad5. Our study also demonstrates that the dose of the Ad vectors has an impact on the memory profile and protective efficacy. The results support further studies with Ad5/3 for malaria vaccine development.

Monitoring Laboratory Occupational Exposures to Burkholderia pseudomallei.

Background: Burkholderia pseudomallei is a Tier 1 overlap select agent and subject to the select agent regulations (42 CFR §73 and 9 CFR §121). It is a gram-negative, motile, soil saprophyte, and the etiologic agent of melioidosis. B. pseudomallei infection can produce systemic illness and can be fatal in the absence of appropriate treatment. Laboratory exposures involving this organism may occur when appropriate containment measures are not employed. Current disease treatment inadequacies and the risk factors associated with melioidosis make this an agent of primary concern in research, commercial, and clinical laboratory environments. Results: This study presents data reported to Centers for Disease Control and Prevention (CDC), Division of Select Agents and Toxins for releases involving B. pseudomallei in 2017-2019 that occurred in Biosafety Level (BSL)-2 and BSL-3 laboratories. Fifty-one Animal and Plant Health Inspection Service (APHIS)/CDC Form 3 release reports led to the medical surveillance of 275 individuals. Entities offered post-exposure prophylaxis to ∼76% of the individuals impacted in the presented events. Summary: Laboratory safety can be improved by implementing appropriate safety precautions to minimize exposures. Most of the incidents discussed in this evidence-based report occurred during work conducted in the absence of primary containment. None of the releases resulted in illness, death, or transmission to or among workers, nor was there transmission outside of a laboratory into the surrounding environment or community. Effective risk assessment and management strategies coupled with standard and special microbiological policies and procedures can result in reduced exposures and improved safety at facilities.

Evaluation of cellular determinants required for in vitro xenotropic murine leukemia virus-related virus entry into human prostate cancer and noncancerous cells.

The newly identified retrovirus-the xenotropic murine leukemia virus-related virus (XMRV)-has recently been shown to be strongly associated with familial prostate cancer in humans (A. Urisman et al., PLoS Pathog. 2:e25, 2006). While that study showed evidence of XMRV infection exclusively in the prostatic stromal fibroblasts, a recent study found XMRV protein antigens mainly in malignant prostate epithelial cells (R. Schlaberg et al., Proc. Natl. Acad. Sci. U. S. A. 106:16351-16356, 2009). To help elucidate the mechanisms behind XMRV infection, we show that prostatic fibroblast cells express Xpr1, a known receptor of XMRV, but its expression is absent in other cell lines of the prostate (i.e., epithelial and stromal smooth muscle cells). We also show that certain amino acid residues located within the predicted extracellular loop (ECL3 and ECL4) sequences of Xpr1 are required for efficient XMRV entry. Although we found strong evidence to support XMRV infection of prostatic fibroblast cell lines via Xpr1, we learned that XMRV was indeed capable of infecting cells that did not necessarily express Xpr1, such as those of the prostatic epithelial and smooth muscle origins. Further studies suggest that the expression of Xpr1 and certain genotypes of the RNASEL gene, which could restrict XMRV infection, may play important roles in defining XMRV tropisms in certain cell types. Collectively, our data reveal important cellular determinants required for XMRV entry into different human prostate cells in vitro, which may provide important insights into the possible role of XMRV as an etiologic agent in human prostate cancer.

Real-time PCR quantitation of subtype C HIV DNA in a Zambian discordant couple cohort.

To study the viral sequence diversity that is characteristic of HIV infection, PCR amplification and sequencing of viral genes is an essential step. However, a limitation of traditional PCR methods is that one viral target may be preferentially amplified over another when multiple sequences are present. This presents a particular problem when conclusions about diversity are made from one or only a few PCRs. One way to avoid resampling is to perform a large number of PCR amplifications on a single template; however, this requires that extensive dilution series be carried out on each patient sample to identify the appropriate concentration of input DNA. Here we describe the development and implementation of a quantitative real-time PCR (qPCR) method that detects a short sequence in gag and is optimized to detect subtype C HIV sequences. The standard curve was externally validated using two chronically infected cell lines carrying a known number of HIV copies per genome, and this assay yielded reproducible and accurate measurements on patient DNA samples over a wide range of input targets. The qPCR assay results were consistent with those obtained by the traditional limiting dilution method yet entailed only a fraction of the time and reagents required for the latter. This robust and quantitative real-time assay can be used to ensure that each viral sequence obtained through PCR represents a single template for studies in which the diversity of the entire population must be accurately portrayed, and can readily be applied to other research settings and viral subtypes.

Substitution of the adenovirus serotype 5 knob with a serotype 3 knob enhances multiple steps in virus replication.

Adenovirus (Ad) serotype 5 (Ad5) continues to be the predominant vector used for cancer gene therapy. However, many tumor types are reported to be relatively refractory to Ad5 infection because of low surface expression of the native Ad5 receptor, CAR. The observation that many tumor cells are CAR deficient has necessitated the development of CAR-independent infection strategies, including the introduction of heterologous ligand sequences into the virus fiber gene and immunological or chemical modifications of the capsid proteins. Alternatively, native Ad5 tropism can be modified by substituting the knob region from other Ad serotypes such as Ad type 3 (Ad3) into the Ad5 knob region. To date, the effect(s) of tropism modification on the replication and oncolytic capacity of these chimeric Ad vectors has not been fully evaluated. To address this issue, Ad5 vectors and isogenically matched chimeric vectors with Ad3 tropism (Ad5/3) were compared in this study. Various parameters of virus infection were compared, including binding, nuclear translocation, E1A transcription, transgene expression, de novo virus production, and oncolysis. Overall, the chimeric Ad5/3 virus was progressively more efficient at each step of the replication cycle compared with its Ad5 counterpart. The higher replication efficiency of the chimeric Ad5/3 vector translated into improved therapeutic efficacy in a murine in vivo tumor rejection model. These findings suggest that in addition to the initial target cell interaction, multiple mechanisms contribute to the enhanced replication of the chimeric Ad5/3 vector. Furthermore, the data demonstrate that alternative Ad serotype receptors can be used to improve infection and subsequent oncolytic replication, which is particularly relevant in gene therapy applications for tumors that are inefficiently infected with Ad5.

Adenoviral gene therapy for renal cancer requires retargeting to alternative cellular receptors.

Metastatic renal cell carcinoma (RCC) is one of the most treatment-resistant malignancies in humans. Therefore, the identification of new agents with better antitumor activity merits a high priority in the treatment of advanced RCC. In this regard, gene therapy with adenoviral (Ad) vectors is a promising new modality for cancer. However, a primary limiting factor for the use of Ad vectors for cancer gene therapy is their critical dependence on cellular expression of the primary Ad receptor, the coxsackie and adenovirus receptor (CAR), known to be down-regulated in many cancer types. Following the identification of CAR deficiency in RCC lines, we have found abundant membrane expression of alpha(v)beta 3 and alpha(v)beta 5 integrins and of the putative receptor to Ad serotype 3 (Ad3). As an alternative gene therapy approach for RCC that would circumvent CAR deficiency, we employed retargeting of replication-incompetent Ad vectors and replication-competent Ad viruses to alpha(v)beta 3 and alpha(v)beta 5 integrins and to the putative Ad3 receptor. These strategies to genetically alter Ad tropism were based on either the insertion of a cysteine-aspartate-cysteine-arginine-glycine-aspartate-cysteine-phenylalanine-cysteine (RGD) motif into the HI loop of the Ad fiber knob domain or on generation of a chimeric Ad fiber composed of adenovirus serotype 5 shaft/Ad3 knob. Both strategies proved highly efficient to circumvent CAR deficiency and enhance gene delivery into RCC cells. Furthermore, in the context of replication-competent Ad, tropism alteration resulted in distinct capacity of the retargeted viruses to infect, replicate, and lyse RCC models in vitro and in vivo. The retargeting strategies were particularly beneficial in the context of replication-competent Ad. These findings underscore the importance of CAR-independent cellular entry mechanisms in RCC and are highly consequential for the development of viral antitumor agents for RCC and other CAR-negative tumors.

Accreditation across cultures: a case study.

Accreditation of academic programs is one of the most significant developments in the evolution of professional education in the United States. Efforts in several fields to extend programmatic accreditation to institutions outside the United States have had mixed results. This report describes such an accreditation experience in health services administration, its pitfalls, and the lessons that the site visit team (the authors) learned. The authors hope that others undertaking such tasks can benefit from this experience.

A model system for the design of armed replicating adenoviruses using p53 as a candidate transgene.

Cancer gene therapy endeavors to overcome the low therapeutic index of currently available therapeutic modalities via the efficient and safe delivery of genetic material into tumor cells. However, despite promising preclinical results, replication-deficient viral vectors have demonstrated a limited efficacy in the clinical setting. To increase vector efficiency, replication-competent viruses have been proposed. Clinical trials have shown the safety of locally injected, conditionally replicative adenoviruses (Ads) but have underscored the need for improved potency. To further increase the therapeutic effect of replicating viral vectors, armed therapeutic viruses (ATVs) have recently been used for high-efficiency transgene expression. However, interference with cellular signaling and viral production by constitutive transgene expression may be counterproductive for ATV replication, thereby hindering the therapeutic outcome. Consequently, studies are equivocal with regard to the potential benefits of ATVs. To address this issue, we hypothesized that induction of replication of an Ad expressing p53 may be a useful strategy in the context of ATV because p53 does not interfere with Ad replication and may even increase its cytolytic effect. We show that in our in vitro ATV model system, E1 transcomplementation of a replication-deficient Ad encoding p53 resulted in dramatic augmentation of cell killing and circumvented resistance to apoptosis. Correlation was found between the degrees of cell killing and apoptosis induction, rather than with viral burst. Furthermore, both Ad5 E1B 55kDa and E4 orf6 genes were required to enhance the cell killing. In conclusion, our p53-ATV model system demonstrates the potential utility of therapeutic transgene expression by a replicating Ad after a rational selection of a candidate transgene.

Replication of an integrin targeted conditionally replicating adenovirus on primary ovarian cancer spheroids.

Replication competent viruses hold promise for treatment of advanced cancers resistant to available therapeutic modalities. Although preliminary clinical results have substantiated their efficacy, preclinical development of these novel approaches is limited by assay substrates. The evaluation of candidate agents could be confounded by differences between primary tumor cells and tumor cell lines, as discordance in the levels of surface receptors relevant for viral entry has been reported. Since primary tumor cells are difficult to analyze ex vivo for longitudinal observation of virus replication, we developed three-dimensional aggregates or spheroids of unpassaged and purified ovarian cancer cells as a means for prolonging primary tumor cell viability and as a three-dimensional in vitro model for replicative viral infection. Ovarian cancer cells purified from ascites samples were sustained for 30 days while retaining the infection profile with tropism modified and unmodified adenoviruses (Ads). Cell line and primary cell spheroids were used to quantitate the replication and oncolytic potency of replicative Ads in preclinical testing for human ovarian cancer trials. Therefore, spheroids provide a method to sustain purified unpassaged primary ovarian cancer cells for extended periods and to allow evaluation of replicative viruses in a three-dimensional model.

CryoEM visualization of an adenovirus capsid-incorporated HIV antigen.

Adenoviral (Ad) vectors show promise as platforms for vaccine applications against infectious diseases including HIV. However, the requirements for eliciting protective neutralizing antibody and cellular immune responses against HIV remain a major challenge. In a novel approach to generate 2F5- and 4E10-like antibodies, we engineered an Ad vector with the HIV membrane proximal ectodomain region (MPER) epitope displayed on the hypervariable region 2 (HVR2) of the viral hexon capsid, instead of expressed as a transgene. The structure and flexibility of MPER epitopes, and the structural context of these epitopes within viral vectors, play important roles in the induced host immune responses. In this regard, understanding the critical factors for epitope presentation would facilitate optimization strategies for developing viral vaccine vectors. Therefore we undertook a cryoEM structural study of this Ad vector, which was previously shown to elicit MPER-specific humoral immune responses. A subnanometer resolution cryoEM structure was analyzed with guided molecular dynamics simulations. Due to the arrangement of hexons within the Ad capsid, there are twelve unique environments for the inserted peptide that lead to a variety of conformations for MPER, including individual α-helices, interacting α-helices, and partially extended forms. This finding is consistent with the known conformational flexibility of MPER. The presence of an extended form, or an induced extended form, is supported by interaction of this vector with the human HIV monoclonal antibody 2F5, which recognizes 14 extended amino acids within MPER. These results demonstrate that the Ad capsid influences epitope structure, flexibility and accessibility, all of which affect the host immune response. In summary, this cryoEM structural study provided a means to visualize an epitope presented on an engineered viral vector and suggested modifications for the next generation of Ad vectors with capsid-incorporated HIV epitopes.

Different patterns of expansion, contraction and memory differentiation of HIV-1 Gag-specific CD8 T cells elicited by adenovirus type 5 and modified vaccinia Ankara vaccines.

The magnitude and functional quality of antiviral CD8 T cell responses are critical for the efficacy of T cell based vaccines. Here, we investigate the influence of two popular viral vectors, adenovirus type 5 (Ad5) and modified vaccinia Ankara (MVA), on expansion, contraction and memory differentiation of HIV-1 Gag insert-specific CD8 T cell responses following immunization and show different patterns for the two recombinant viral vectors. The Ad5 vector primed 6-fold higher levels of insert-specific CD8 effector T cells than the MVA vector. The Ad5-primed effector cells also underwent less contraction (<2-fold) than the MVA-primed cells (>5-fold). The Ad5-primed memory cells were predominantly CD62L negative (effector memory) whereas the MVA-primed memory cells were predominantly CD62L positive (central memory). Consistent with their memory phenotype, MVA-primed CD8 T cells underwent higher fold expansion than Ad5-primed CD8 T cells following a homologous or heterologous boost. Impressively, the Ad5 boost changed the quality of MVA-primed memory response such that they undergo less contraction with effector memory phenotype. However, the MVA boost did not influence the contraction and memory phenotype of Ad5-primed response. In conclusion, our results demonstrate that vaccine vector strongly influences the expansion, contraction and the functional quality of insert-specific CD8 T cell responses and have implications for vaccine development against infectious diseases.

Antibody responses against xenotropic murine leukemia virus-related virus envelope in a murine model.

BACKGROUND: Xenotropic murine leukemia virus-related virus (XMRV) was recently discovered to be the first human gammaretrovirus that is associated with chronic fatigue syndrome and prostate cancer (PC). Although a mechanism for XMRV carcinogenesis is yet to be established, this virus belongs to the family of gammaretroviruses well known for their ability to induce cancer in the infected hosts. Since its original identification XMRV has been detected in several independent investigations; however, at this time significant controversy remains regarding reports of XMRV detection/prevalence in other cohorts and cell type/tissue distribution. The potential risk of human infection, coupled with the lack of knowledge about the basic biology of XMRV, warrants further research, including investigation of adaptive immune responses. To study immunogenicity in vivo, we vaccinated mice with a combination of recombinant vectors expressing codon-optimized sequences of XMRV gag and env genes and virus-like particles (VLP) that had the size and morphology of live infectious XMRV. RESULTS: Immunization elicited Env-specific binding and neutralizing antibodies (NAb) against XMRV in mice. The peak titers for ELISA-binding antibodies and NAb were 1:1024 and 1:464, respectively; however, high ELISA-binding and NAb titers were not sustained and persisted for less than three weeks after immunizations. CONCLUSIONS: Vaccine-induced XMRV Env antibody titers were transiently high, but their duration was short. The relatively rapid diminution in antibody levels may in part explain the differing prevalences reported for XMRV in various prostate cancer and chronic fatigue syndrome cohorts. The low level of immunogenicity observed in the present study may be characteristic of a natural XMRV infection in humans.

HIV antigen incorporation within adenovirus hexon hypervariable 2 for a novel HIV vaccine approach.

Adenoviral (Ad) vectors have been used for a variety of vaccine applications including cancer and infectious diseases. Traditionally, Ad-based vaccines are designed to express antigens through transgene expression of a given antigen. However, in some cases these conventional Ad-based vaccines have had sub-optimal clinical results. These sub-optimal results are attributed in part to pre-existing Ad serotype 5 (Ad5) immunity. In order to circumvent the need for antigen expression via transgene incorporation, the "antigen capsid-incorporation" strategy has been developed and used for Ad-based vaccine development in the context of a few diseases. This strategy embodies the incorporation of antigenic peptides within the capsid structure of viral vectors. The major capsid protein hexon has been utilized for these capsid incorporation strategies due to hexon's natural role in the generation of anti-Ad immune response and its numerical representation within the Ad virion. Using this strategy, we have developed the means to incorporate heterologous peptide epitopes specifically within the major surface-exposed domains of the Ad capsid protein hexon. Our study herein focuses on generation of multivalent vaccine vectors presenting HIV antigens within the Ad capsid protein hexon, as well as expressing an HIV antigen as a transgene. These novel vectors utilize HVR2 as an incorporation site for a twenty-four amino acid region of the HIV membrane proximal ectodomain region (MPER), derived from HIV glycoprotein gp41 (gp41). Our study herein illustrates that our multivalent anti-HIV vectors elicit a cellular anti-HIV response. Furthermore, vaccinations with these vectors, which present HIV antigens at HVR2, elicit a HIV epitope-specific humoral immune response.

XMRV infection in patients with prostate cancer: novel serologic assay and correlation with PCR and FISH.

OBJECTIVES: To develop a serum-based assay to detect neutralizing antibodies to the xenotropic murine leukemia virus-related virus (XMRV) retrovirus and to use this assay with polymerase chain reaction and fluorescence in situ hybridization to identify patients with prostate cancer previously exposed to XMRV infection and those who carry XMRV viral sequences in their prostate. METHODS: Patients who had undergone radical prostatectomy were enrolled, and biologic specimens were obtained at surgery. The patients were genotyped for the R462Q RNASEL variant using a TaqMan genotyping assay on DNA from the peripheral blood. A serum assay that detects XMRV neutralizing antibodies was developed and used to determine which patients had serologic evidence of previous infection with XMRV virus. Some of these patients were also tested for the presence of XMRV nucleotide sequences in their prostate using polymerase chain reaction and fluorescence in situ hybridization analysis. RESULTS: At a serum dilution of 1:150, our assay detected 11 (27.5%) of 40 patients with XMRV neutralizing antibodies, including 8 (40%) of 20 with the RNASEL genotype QQ and 3 (15%) of 20 with either the RQ or RR genotype. These results were in complete concordance with 2 other assays (polymerase chain reaction and fluorescence in situ hybridization), which were designed to detect XMRV infection. CONCLUSIONS: XMRV infects some patients with prostate cancer. Neutralizing antibodies against XMRV correlated with 2 independent methods of detecting the virus in the prostate. The antibody response suggests that with clinical serologic assay development, it might be possible to screen patients for XMRV infection. The cases presented in the present report provided biologic samples that can be used for the development of a clinically relevant assay.

Lac-regulated system for generating adenovirus 5 vaccine vectors expressing cytolytic human immunodeficiency virus 1 genes.

Adenovirus (Ad) vectors have been developed as human immunodeficiency-1 (HIV-1) vaccine vectors because they consistently induce immune responses in preclinical animal models and human trials. Strong promoters and codon-optimization are often used to enhance vaccine-induced HIV-1 gene expression and immunogenicity. However, if the transgene is inherently cytotoxic in the cell line used to produce the vector, and is expressed at high levels, it is difficult to rescue a stable Ad HIV-1 vaccine vector. Therefore we hypothesized that generation of Ad vaccine vectors expressing cytotoxic genes, such as HIV-1 env, would be more efficient if expression of the transgene was down-regulated during Ad rescue. To test this hypothesis, a Lac repressor-operator system was applied to regulate expression of reporter luciferase and HIV-1 env transgenes during Ad rescue. The results demonstrate that during Ad rescue, constitutive expression of the Lac repressor in 293 cells reduced transgene expression levels to approximately 5% of that observed in the absence of regulation. Furthermore, Lac-regulation translated into more efficient Ad rescue compared to traditional 293 cells. Importantly, Ad vectors rescued with this system showed high levels of transgene expression when transduced into cells that lack the Lac repressor protein. The Lac-regulated system also facilitated the rescue of modified Ad vectors that have non-native receptor tropism. These tropism-modified Ad vectors infect a broader range of cell types than the unmodified Ad, which could increase their effectiveness as a vaccine vector. Overall, the Lac-regulated system described here (i) is backwards compatible with Ad vector methods that employ bacterial-mediated homologous recombination, (ii) is adaptable for the engineering of tropism-modified Ad vectors, and (iii) does not require co-expression of regulatory genes from the vector or the addition of exogenous chemicals to induce or repress transgene expression. This system therefore could facilitate the development of Ad-based vaccine candidates that otherwise would not be feasible to generate.

Interaction between the cellular protein eEF1A and the 3'-terminal stem-loop of West Nile virus genomic RNA facilitates viral minus-strand RNA synthesis.

RNase footprinting and nitrocellulose filter binding assays were previously used to map one major and two minor binding sites for the cell protein eEF1A on the 3'(+) stem-loop (SL) RNA of West Nile virus (WNV) (3). Base substitutions in the major eEF1A binding site or adjacent areas of the 3'(+) SL were engineered into a WNV infectious clone. Mutations that decreased, as well as ones that increased, eEF1A binding in in vitro assays had a negative effect on viral growth. None of these mutations affected the efficiency of translation of the viral polyprotein from the genomic RNA, but all of the mutations that decreased in vitro eEF1A binding to the 3' SL RNA also decreased viral minus-strand RNA synthesis in transfected cells. Also, a mutation that increased the efficiency of eEF1A binding to the 3' SL RNA increased minus-strand RNA synthesis in transfected cells, which resulted in decreased synthesis of genomic RNA. These results strongly suggest that the interaction between eEF1A and the WNV 3' SL facilitates viral minus-strand synthesis. eEF1A colocalized with viral replication complexes (RC) in infected cells and antibody to eEF1A coimmunoprecipitated viral RC proteins, suggesting that eEF1A facilitates an interaction between the 3' end of the genome and the RC. eEF1A bound with similar efficiencies to the 3'-terminal SL RNAs of four divergent flaviviruses, including a tick-borne flavivirus, and colocalized with dengue virus RC in infected cells. These results suggest that eEF1A plays a similar role in RNA replication for all flaviviruses.

Role of V1V2 and other human immunodeficiency virus type 1 envelope domains in resistance to autologous neutralization during clade C infection.

Biologically functional clade C envelope (Env) glycoproteins from the chronically (donor) and newly (recipient) infected partners of four heterosexual transmission pairs in Zambia were cloned and characterized previously. In each case, the donor viral quasispecies contained Envs that were resistant to autologous neutralization by contemporaneous plasma, while the recipient Envs were sensitive to neutralizing antibodies in this donor plasma sample. The donor Envs also varied in length, glycosylation, and amino acid sequence of the V1V2 hypervariable domain of gp120, while the recipient Envs were much more homogeneous. To assess the contribution of V1V2 to the neutralization phenotype of the donor Envs, V1V2 domains from neutralization-sensitive recipient Envs were replaced with donor V1V2 domains, and the autologous neutralization sensitivities of the chimeric Envs were evaluated using a virus-pseudotyping assay. Long donor V1V2 domains regulated sensitivity to autologous neutralization, although the effect was dependent on the Env background. Short donor V1V2 domains did not confer neutralization resistance. Primary sequence differences in V2 were also found to influence neutralization sensitivity in one set of recipient Envs. The results demonstrate that expansion of the V1V2 domain is one pathway to escape from autologous neutralization in subtype C Envs. However, V1V2-independent mechanisms of resistance also exist, suggesting that escape is multifaceted in chronic subtype C infection.

Unique mutational patterns in the envelope alpha 2 amphipathic helix and acquisition of length in gp120 hypervariable domains are associated with resistance to autologous neutralization of subtype C human immunodeficiency virus type 1.

Autologous neutralizing antibodies (NAb) against human immunodeficiency virus type 1 generate viral escape variants; however, the mechanisms of escape are not clearly defined. In a previous study, we determined the susceptibilities of 48 donor and 25 recipient envelope (Env) glycoproteins from five subtype C heterosexual transmission pairs to NAb in donor plasma by using a virus pseudotyping assay, thereby providing an ideal setting to probe the determinants of susceptibility to neutralization. In the present study, acquisition of length in the Env gp120 hypervariable domains was shown to correlate with resistance to NAb in donor plasma (P = 0.01; Kendall's tau test) but not in heterologous plasma. Sequence divergence in the gp120 V1-to-V4 region also correlated with resistance to donor (P = 0.0002) and heterologous (P = 0.001) NAb. A mutual information analysis suggested possible associations of nine amino acid positions in V1 to V4 with NAb resistance to the donor's antibodies, and five of these were located within an 18-residue amphipathic helix (alpha2) located on the gp120 outer domain. High nonsynonymous-to-synonymous substitution (dN/dS) ratios, indicative of positive selection, were also found at these five positions in subtype C sequences in the database. Nevertheless, exchange of the entire alpha2 helix between resistant donor Envs and sensitive recipient Envs did not alter the NAb phenotype. The combined mutual information and dN/dS analyses suggest that unique mutational patterns in alpha2 and insertions in the V1-to-V4 region are associated with NAb resistance during subtype C infection but that the selected positions within the alpha2 helix must be linked to still other changes in Env to confer antibody escape. These findings suggest that subtype C viruses utilize mutations in the alpha2 helix for efficient viral replication and immune avoidance.

Evidence for potent autologous neutralizing antibody titers and compact envelopes in early infection with subtype C human immunodeficiency virus type 1.

Information about neutralizing antibody responses in subtype C-infected individuals is limited, even though this viral subtype causes the majority of AIDS cases worldwide. Here we compared the course and magnitude of the autologous neutralizing antibody (NAb) response against viral envelope (Env) glycoproteins present during acute and early infection with subtypes B and C human immunodeficiency virus type 1 (HIV-1). NAb responses were evaluated in 6 subtype B-infected and 11 subtype C-infected subjects over a mean evaluation period of 25 months using a pseudovirus reporter gene assay. All subjects in the C cohort were infected through heterosexual contact, while five of the six subjects in the B cohort were infected via male-to-male contact. The kinetics and magnitude of the NAb responses varied among subjects in the B and C cohorts; however, the median 50% inhibitory concentration (IC(50) titer) reached by antibody in the plasma of subtype C-infected subjects, overall, was 3.5-fold higher than in the subtype B-infected subjects (P = 0.06). The higher titers of NAbs in the C cohort were associated with viruses having significantly shorter amino acid length (P = 0.002) in the V1 to V4 region of the surface Env glycoprotein, gp120, compared to the B cohort. Despite the potency of the autologous subtype C NAb response, it was not directed against cross-neutralizing epitopes. These data demonstrate that subtype C Envs elicit a potent yet restricted NAb response early in infection that frequently reaches IC(50) titers in excess of 1:1,000 and suggest that clade-specific differences may exist in Env immunogenicity or susceptibility to neutralization.