Victoria R. Polonis

Human Immunodeficiency Virus Type 1 env Clones from Acute and Early Subtype B Infections for Standardized Assessments of Vaccine-Elicited Neutralizing Antibodies

ABSTRACT Induction of broadly cross-reactive neutralizing antibodies is a high priority for AIDS vaccine development but one that has proven difficult to be achieved. While most immunogens generate antibodies that neutralize a subset of T-cell-line-adapted strains of human immunodeficiency virus type 1 (HIV-1), none so far have generated a potent, broadly cross-reactive response against primary isolates of the virus. Even small increments in immunogen improvement leading to increases in neutralizing antibody titers and cross-neutralizing activity would accelerate vaccine development; however, a lack of uniformity in target strains used by different investigators to assess cross-neutralization has made the comparison of vaccine-induced antibody responses difficult. Thus, there is an urgent need to establish standard panels of HIV-1 reference strains for wide distribution. To facilitate this, full-length gp160 genes were cloned from acute and early subtype B infections and characterized for use as reference reagents to assess neutralizing antibodies against clade B HIV-1. Individual gp160 clones were screened for infectivity as Env-pseudotyped viruses in a luciferase reporter gene assay in JC53-BL (TZM-bl) cells. Functional env clones were sequenced and their neutralization phenotypes characterized by using soluble CD4, monoclonal antibodies, and serum samples from infected individuals and noninfected recipients of a recombinant gp120 vaccine. Env clones from 12 R5 primary HIV-1 isolates were selected that were not unusually sensitive or resistant to neutralization and comprised a wide spectrum of genetic, antigenic, and geographic diversity. These reference reagents will facilitate proficiency testing and other validation efforts aimed at improving assay performance across laboratories and can be used for standardized assessments of vaccine-elicited neutralizing antibodies.

Tiered Categorization of a Diverse Panel of HIV-1 Env Pseudoviruses for Assessment of Neutralizing Antibodies

ABSTRACT The restricted neutralization breadth of vaccine-elicited antibodies is a major limitation of current human immunodeficiency virus-1 (HIV-1) candidate vaccines. In order to permit the efficient identification of vaccines with enhanced capacity for eliciting cross-reactive neutralizing antibodies (NAbs) and to assess the overall breadth and potency of vaccine-elicited NAb reactivity, we assembled a panel of 109 molecularly cloned HIV-1 Env pseudoviruses representing a broad range of genetic and geographic diversity. Viral isolates from all major circulating genetic subtypes were included, as were viruses derived shortly after transmission and during the early and chronic stages of infection. We assembled a panel of genetically diverse HIV-1-positive (HIV-1+) plasma pools to assess the neutralization sensitivities of the entire virus panel. When the viruses were rank ordered according to the average sensitivity to neutralization by the HIV-1+ plasmas, a continuum of average sensitivity was observed. Clustering analysis of the patterns of sensitivity defined four subgroups of viruses: those having very high (tier 1A), above-average (tier 1B), moderate (tier 2), or low (tier 3) sensitivity to antibody-mediated neutralization. We also investigated potential associations between characteristics of the viral isolates (clade, stage of infection, and source of virus) and sensitivity to NAb. In particular, higher levels of NAb activity were observed when the virus and plasma pool were matched in clade. These data provide the first systematic assessment of the overall neutralization sensitivities of a genetically and geographically diverse panel of circulating HIV-1 strains. These reference viruses can facilitate the systematic characterization of NAb responses elicited by candidate vaccine immunogens.

Genetic and Neutralization Properties of Subtype C Human Immunodeficiency Virus Type 1 Molecular env Clones from Acute and Early Heterosexually Acquired Infections in Southern Africa

ABSTRACT A standard panel of subtype C human immunodeficiency virus type 1 (HIV-1) Env-pseudotyped viruses was created by cloning, sequencing, and characterizing functional gp160 genes from 18 acute and early heterosexually acquired infections in South Africa and Zambia. In general, the gp120 region of these clones was shorter (most evident in V1 and V4) and less glycosylated compared to newly transmitted subtype B viruses, and it was underglycosylated but no different in length compared to chronic subtype C viruses. The gp120s also exhibited low amino acid sequence variability (12%) in V3 and high variability (39%) immediately downstream of V3, a feature shared with newly transmitted subtype B viruses and chronic viruses of both subtypes. When tested as Env-pseudotyped viruses in a luciferase reporter gene assay, all clones possessed an R5 phenotype and resembled primary isolates in their sensitivity to neutralization by HIV-1-positive plasmas. Results obtained with a multisubtype plasma panel suggested partial subtype preference in the neutralizing antibody response to infection. The clones were typical of subtype C in that all were resistant to 2G12 (associated with loss of N-glycosylation at position 295) and most were resistant to 2F5, but all were sensitive to 4E10 and many were sensitive to immunoglobulin G1b12. Finally, conserved neutralization epitopes in the CD4-induced coreceptor binding domain of gp120 were poorly accessible and were difficult to induce and stabilize with soluble CD4 on Env-pseudotyped viruses. These results illustrate key genetic and antigenic properties of subtype C HIV-1 that might impact the design and testing of candidate vaccines. A subset of these gp160 clones are suitable for use as reference reagents to facilitate standardized assessments of vaccine-elicited neutralizing antibody responses.

Recommendations for the Design and Use of Standard Virus Panels To Assess Neutralizing Antibody Responses Elicited by Candidate Human Immunodeficiency Virus Type 1 Vaccines

Laboratory measures of antigen-specific immunity are an essential component of the vaccine discovery process. For human immunodeficiency virus type 1 (HIV-1), this process will likely require iterative evaluations of vaccine immunogens to choose the most promising vaccine candidates to advance into human trials. To optimally evaluate and compare vaccine immunogens, we will need high-throughput assays that allow accurate and reproducible measurements of immune responses. In addition, vaccine sponsors and regulatory agencies appropriately require that immune assays associated with human trials be performed in laboratories that comply with guidelines for good laboratory practices (GLP). The rigorous process of assay validation associated with GLP can improve the accuracy of immune assessment assays and contribute to vaccine development by advancing our ability to distinguish incremental improvements in immune responses elicited by novel immunogens. In this commentary, we address several of these issues with regard to the measurement of anti-HIV-1 neutralizing antibodies (NAbs). We recommend the use of DNA plasmids encoding full-length functional HIV-1 envelope glycoproteins (Env); these env clones, when transfected with an HIV-1 envdefective molecular clone, produce well-characterized HIV-1 Env pseudovirions. Additionally, we recommend the establishment of standardized panels of Env-pseudotyped viruses to assess the potencies and breadths of NAbs elicited by vaccine immunogens. These virus panels would form the basis for GLP neutralization assays used to assess sera from clinical vaccine studies, and the same virus panels could be used by investigators interested in the preclinical evaluation of vaccine immunogens.

Magnitude and Breadth of the Neutralizing Antibody Response in the RV144 and Vax003 HIV-1 Vaccine Efficacy Trials

Background. A recombinant canarypox vector expressing human immunodeficiency virus type 1 (HIV-1) Gag, Pro, and membrane-linked gp120 (vCP1521), combined with a bivalent gp120 protein boost (AIDSVAX B/E), provided modest protection against HIV-1 infection in a community-based population in Thailand (RV144 trial). No protection was observed in Thai injection drug users who received AIDSVAX B/E alone (Vax003 trial). We compared the neutralizing antibody response in these 2 trials. Methods. Neutralization was assessed with tier 1 and tier 2 strains of virus in TZM-bl and A3R5 cells. Results. Neutralization of several tier 1 viruses was detected in both RV144 and Vax003. Peak titers were higher in Vax003 and waned rapidly in both trials. The response in RV144 was targeted in part to V3 of gp120.vCP1521 priming plus 2 boosts with gp120 protein was superior to 2 gp120 protein inoculations alone, confirming a priming effect for vCP1521. Sporadic weak neutralization of tier 2 viruses was detected only in Vax003 and A3R5 cells. Conclusion. The results suggest either that weak neutralizing antibody responses can be partially protective against HIV-1 in low-risk heterosexual populations or that the modest efficacy seen in RV144 was mediated by other immune responses, either alone or in combination with neutralizing antibodies.

A Phase I Evaluation of the Safety and Immunogenicity of Vaccination with Recombinant gp160 in Patients with Early Human Immunodeficiency Virus Infection

BACKGROUND Despite multiple antiviral humoral and cellular immune responses, infection with the human immunodeficiency virus (HIV) results in a progressively debilitating disease. We hypothesized that a more effective immune response could be generated by post-infection vaccination with HIV-specific antigens. METHODS We performed a phase I trial of the safety and immunogenicity of a vaccine prepared from molecularly cloned envelope protein, gp160, in 30 volunteer subjects with HIV infection in Walter Reed stage 1 or 2. The vaccine was administered either on days 0, 30, and 120 or on days 0, 30, 60, 120, 150, and 180. HIV-specific humoral and cellular immune responses were measured; local and systemic reactions to vaccination, including general measures of immune function, were monitored. RESULTS In 19 of the 30 subjects both humoral and cellular immunity to HIV envelope proteins increased in response to vaccination with gp160. Seroconversion to selected envelope epitopes was observed, as were new T-cell proliferative responses to gp160. Response was associated with the CD4 cell count determined before vaccination (13 of 16 subjects [81 percent] with greater than 600 cells per milliliter responded, as compared with 6 of 14 [43 percent] with less than or equal to 600 cells per milliliter; P = 0.07) and with the number of injections administered (87 percent of subjects randomly assigned to receive six injections responded, as compared with 40 percent of those assigned to three injections; P = 0.02). Local reactions at the site of injection were mild. There were no adverse systemic reactions, including diminution of general in vitro or in vivo cellular immune function. After 10 months of follow-up, the mean CD4 count had not decreased in the 19 subjects who responded, but it had decreased by 7.3 percent in the 11 who did not respond. CONCLUSIONS This gp160 vaccine is safe and immunogenic in volunteer patients with early HIV infection. Although it is too early to know whether this approach will be clinically useful, further scientific and therapeutic evaluation of HIV-specific vaccine therapy is warranted. Similar vaccines may prove to be effective for other chronic infections.

Human Monoclonal Antibodies Specific for Conformation-Sensitive Epitopes of V3 Neutralize Human Immunodeficiency Virus Type 1 Primary Isolates from Various Clades

ABSTRACT The epitopes of the V3 domain of the human immunodeficiency virus type 1 (HIV-1) gp120 glycoprotein have complex structures consisting of linear and conformational antigenic determinants. Anti-V3 antibodies (Abs) recognize both types of elements, but Abs which preferentially react to the conformational aspect of the epitopes may have more potent neutralizing activity against HIV-1, as recently suggested. To test this hypothesis, human anti-V3 monoclonal Abs (MAbs) were selected using a V3 fusion protein (V3-FP) which retains the conformation of the third variable region. The V3-FP consists of the V3JR-CSF sequence inserted into a truncated form of murine leukemia virus gp70. Six human MAbs which recognize epitopes at the crown of the V3 loop were selected with the V3-FP. They were found to react more strongly with molecules displaying conformationally intact V3 than with linear V3 peptides. In a virus capture assay, these MAbs showed cross-clade binding to native, intact virions of clades A, B, C, D, and F. No binding was found to isolates from subtype E. The neutralizing activity of MAbs against primary isolates was determined in three assays: the GHOST cell assay, a phytohemagglutinin-stimulated peripheral blood mononuclear cell assay, and a luciferase assay. While these new MAbs displayed various degrees of activity, the pattern of cross-clade neutralization of clades A, B, and F was most pronounced. The neutralization of clades C and D viruses was weak and sporadic, and neutralization of clade E by these MAbs was not detected. Analysis by linear regression showed a highly significant correlation (P < 0.0001) between the strength of binding of these anti-V3 MAbs to intact virions and the percent neutralization. These studies demonstrate that human MAbs to conformation-sensitive epitopes of V3 display cross-clade reactivity in both binding to native, intact virions and neutralization of primary isolates.

Replication Competent Molecular Clones of HIV-1 Expressing Renilla Luciferase Facilitate the Analysis of Antibody Inhibition in PBMC

Effective vaccine development for human immunodeficiency virus type 1 (HIV-1) will require assays that ascertain the capacity of vaccine immunogens to elicit neutralizing antibodies (NAb) to diverse HIV-1 strains. To facilitate NAb assessment in peripheral blood mononuclear cell (PBMC)-based assays, we developed an assay-adaptable platform based on a Renilla luciferase (LucR) expressing HIV-1 proviral backbone. LucR was inserted into pNL4-3 DNA, preserving all viral open reading frames. The proviral genome was engineered to facilitate expression of diverse HIV-1 env sequences, allowing analysis in an isogenic background. The resulting Env-IMC-LucR viruses are infectious, and LucR is stably expressed over multiple replications in PBMC. HIV-1 neutralization, targeting TZM-bl cells, was highly correlative comparing virus (LucR) and cell (firefly luciferase) readouts. In PBMC, NAb activity can be analyzed either within a single or multiple cycles of replication. These results represent advancement toward a standardizable PBMC-based neutralization assay for assessing HIV-1 vaccine immunogen efficacy.

Safety and immunogenicity of an HIV subtype B and E prime-boost vaccine combination in HIV-negative Thai adults

ALVAC-HIV (vCP1521) and AIDSVAX B/E were evaluated in a phase 1/2 trial of human immunodeficiency virus (HIV)-negative Thai adults. Of 133 volunteers enrolled, 122 completed the trial. There were no serious vaccine-related adverse events, nor were there intercurrent HIV infections. Lymphoproliferative responses to glycoprotein 120 E were induced in 63% of the volunteers, and HIV-specific CD8 cytotoxic T lymphocyte responses were induced in 24%. Antibody responses increased in frequency and magnitude in association with the dose level of AIDSVAX B/E. Binding and neutralizing antibodies to the MN strain were induced in 100% and 98%, respectively, of the volunteers receiving 600 microg of AIDSVAX B/E, and such antibodies to E strains were induced in 96% and 71%, respectively, of these volunteers. This vaccine combination was well tolerated and was immunogenic, meeting milestones for advancement to phase 3 evaluation.

Biologic and Genetic Characterization of a Panel of 60 Human Immunodeficiency Virus Type 1 Isolates, Representing Clades A, B, C, D, CRF01_AE, and CRF02_AG, for the Development and Assessment of Candidate Vaccines

ABSTRACT A critical priority for human immunodeficiency virus type 1 (HIV-1) vaccine development is standardization of reagents and assays for evaluation of immune responses elicited by candidate vaccines. To provide a panel of viral reagents from multiple vaccine trial sites, 60 international HIV-1 isolates were expanded in peripheral blood mononuclear cells and characterized both genetically and biologically. Ten isolates each from clades A, B, C, and D and 10 isolates each from CRF01_AE and CRF02_AG were prepared from individuals whose HIV-1 infection was evaluated by complete genome sequencing. The main criterion for selection was that the candidate isolate was pure clade or pure circulating recombinant. After expansion in culture, the complete envelope (gp160) of each isolate was verified by sequencing. The 50% tissue culture infectious dose and p24 antigen concentration for each viral stock were determined; no correlation between these two biologic parameters was found. Syncytium formation in MT-2 cells and CCR5 or CXCR4 coreceptor usage were determined for all isolates. Isolates were also screened for neutralization by soluble CD4, a cocktail of monoclonal antibodies, and a pool of HIV-1-positive patient sera. The panel consists of 49 nonsyncytium-inducing isolates that use CCR5 as a major coreceptor and 11 syncytium-inducing isolates that use only CXCR4 or both coreceptors. Neutralization profiles suggest that the panel contains both neutralization-sensitive and -resistant isolates. This collection of HIV-1 isolates represents the six major globally prevalent strains, is exceptionally large and well characterized, and provides an important resource for standardization of immunogenicity assessment in HIV-1 vaccine trials.