Safety, pharmacokinetics and neutralization of the broadly neutralizing HIV-1 human monoclonal antibody VRC01 in healthy adults.

VRC-HIVMAB060-00-AB (VRC01) is a broadly neutralizing HIV-1 monoclonal antibody (mAb) isolated from the B cells of an HIV-infected patient. It is directed against the HIV-1 CD4 binding site and is capable of potently neutralizing the majority of diverse HIV-1 strains. This Phase I dose-escalation study in healthy adults was conducted at the National Institutes of Health (NIH) Clinical Center (Bethesda, MD, USA). Primary objectives were the safety, tolerability and pharmacokinetics (PK) of VRC01 intravenous (i.v.) infusion at 5, 20 or 40 mg/kg, given either once (20 mg/kg) or twice 28 days apart (all doses), and of subcutaneous (s.c.) delivery at 5 mg/kg compared to s.c. placebo given twice, 28 days apart. Cumulatively, 28 subjects received 43 VRC01 and nine received placebo administrations. There were no serious adverse events or dose-limiting toxicities. Mean 28-day serum trough concentrations after the first infusion were 35 and 57 µg/ml for groups infused with 20 mg/kg (n = 8) and 40 mg/kg (n = 5) doses, respectively. Mean 28-day trough concentrations after the second infusion were 56 and 89 µg/ml for the same two doses. Over the 5-40 mg/kg i.v. dose range (n = 18), the clearance was 0.016 l/h and terminal half-life was 15 days. After infusion VRC01 retained expected neutralizing activity in serum, and anti-VRC01 antibody responses were not detected. The human monoclonal antibody (mAb) VRC01 was well tolerated when delivered i.v. or s.c. The mAb demonstrated expected half-life and pharmacokinetics for a human immunoglobulin G. The safety and PK results support and inform VRC01 dosing schedules for planning HIV-1 prevention efficacy studies.

Passive immunization with a human monoclonal antibody protects hu-PBL-SCID mice against challenge by primary isolates of HIV-1.

How well antibodies can protect against disease due to HIV-1 infection remains a pivotal but unresolved issue with important implications for vaccine design and the use of prophylactic antibody to prevent infection after accidental exposure to the virus and to interrupt transmission of virus from mother to child. Strong doubts about the possible utility of antibodies in vivo have been raised because of the relative resistance of primary viruses to antibody neutralization in vitro. Primary viruses are likely to be close to the viruses transmitted during natural infection in humans. Vaccine studies have been of little value in assessing antibody efficacy in vivo because none of the strategies described to date have elicited significant neutralizing antibody responses to primary viruses. Passive immunization studies are similarly hindered by the paucity of reagents able to neutralize primary viruses effectively and a single study has suggested some benefit. Here we describe experiments to explore the ability of passive antibody to protect against primary virus challenge in hu-PBL-SCID mice. In this model, severe combined immunodeficient (SCID) mice are populated with human peripheral blood mononuclear cells (PBMCs) and infected with HIV-1. We find that the potent neutralizing human monoclonal antibody IgG1b12 at high dose is able to completely protect even when given several hours after viral challenge. The results are encouraging for antibody-based postexposure prophylaxis and support the notion that antibody induction could contribute to an effective vaccine.

HIV-1-specific CD4+ T cells are detectable in most individuals with active HIV-1 infection, but decline with prolonged viral suppression.

The role of HIV-1-specific CD4+ T-cell responses in controlling HIV-1 infection remains unclear. Previous work has suggested that such cells are eliminated in the early stages of infection in most subjects, and thus cannot substantially contribute to host defense against HIV-1. Here, using flow cytometric detection of antigen-induced intracellular cytokines, we show that significant frequencies of gag specific, T-helper-1 CD4+ memory T cells are detectable in most subjects with active/progressive HIV-1 infection (median frequency, 0.12% of memory subset; range, 0-0.66%). Median frequencies of these cells were considerably higher in nonprogressive HIV-1 disease (0.40%), but there was substantial overlap between the two groups (range of nonprogressors, 0.10-1.7%). Continuous HIV-1 suppression with anti-retroviral therapy was associated with a time-dependent reduction in median frequencies of gag-specific CD4+ memory T cells: 0.08% in subjects treated for 4-24 weeks, and 0.03% in subjects treated for 47-112 weeks. Thus, functional HIV-1-specific CD4+ T cells are commonly available for support of anti-HIV-1 effector responses in active disease, but their decline with anti-retroviral therapy indicates that immunologic participation in long-term HIV-1 control will probably require effective vaccination strategies.

Maternal HIV-1 viral load and vertical transmission of infection: the Ariel Project for the prevention of HIV transmission from mother to infant.

Most HIV-1 infections of children result from mother-to-infant transmission, which may occur perinatally or postnatally, as a consequence of breast feeding. In this study, the influence of maternal viral load on transmission of infection to infants from non-breast-feeding mothers was examined using samples of plasma and peripheral blood mononuclear cells (PBMCs) collected at several time points during pregnancy and the 6-month period after delivery. These samples were analyzed by several quantitative methods, including virus cultures of PBMCs and polymerase chain reaction (PCR) assays for HIV-1 RNA in plasma and DNA in PBMCs. The risk of transmission increased slightly with a higher viral load, but transmission and nontransmission occurred over the entire range of values for each assay. No threshold value of virus load was identified which discriminated between transmitters and nontransmitters. We also noted a significant rise in viral load and a decline in CD4+ lymphocytes in the six months after delivery. These findings suggest that a high maternal viral load is insufficient to fully explain vertical transmission of HIV-1. Additional studies are needed to examine the post-partum increase in viremia.

Relative resistance to HIV-1 infection of CD4 lymphocytes from persons who remain uninfected despite multiple high-risk sexual exposure.

Some individuals remain uninfected with human immunodeficiency virus type-1 (HIV-1) despite multiple high-risk sexual exposures. We studied a cohort of 25 subjects with histories of multiple high-risk sexual exposures to HIV-1 and found that their CD8+ lymphocytes had greater anti-HIV-1 activity than did CD8+ lymphocytes from nonexposed controls. Further studies indicated that their purified CD4+ lymphocytes were less susceptible to infection with multiple primary isolates of HIV-1 than were CD4+ lymphocytes from the nonexposed controls. This relative resistance to HIV-1 infection did not extend to T-cell line-adapted strains, was restricted by the envelope glycoprotein, was not explained by the cell surface density of CD4 molecules, but was associated with the activity of the C-C chemokines RANTES, MIP-1alpha, and MIP-1beta. This relative resistance of CD4+ lymphocytes may contribute to protection from HIV-1 in multiply exposed persons.

The role of a mutant CCR5 allele in HIV-1 transmission and disease progression.

A 32-nucleotide deletion (delta 32) within the beta-chemokine receptor 5 (CCR5) gene has been described in subjects who remain uninfected despite extensive exposure to HIV-1. This allele was found to be common in the Caucasian population with a frequency of 0.0808, but was not found in people of African or Asian ancestry. To determine its role in HIV-1 transmission and disease progression, we analyzed the CCRS genotype of 1252 homosexual men enrolled in the Chicago component of the Multicenter AIDS Cohort Study (MACS). No infected participant was found to be homozygous for the delta 32 allele, whereas 3.6% of at-risk but uninfected Caucasian participants were homozygous, showing the highly protective role of this genotype against sexual acquisition of HIV-1. No evidence was found to suggest that heterozygotes were protected against HIV-1 infection, but a limited protective role against disease progression was noted. The delta 32 allele of CCR5 is therefore an important host factor in HIV-1 transmission and pathogenesis.

Characterization of human immunodeficiency virus type 1-specific cytotoxic T lymphocyte clones isolated during acute seroconversion: recognition of autologous virus sequences within a conserved immunodominant epitope.

Virus-specific cytotoxic T lymphocytes (CTL) are involved in protective immunity to many virus infections. It has recently been shown that CTL are detectable early during primary infection with the primate lentiviruses, human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus. To better characterize the CTL response during acute HIV-1 infection, HIV-1-specific CTL clones were generated from two patients during symptomatic HIV-1 seroconversion. These CTL clones demonstrated specificity for env of HIV-1 and recognized sequences within gp41. Two human histocompatibility leukocyte antigen (HLA) A31-restricted clones from the same individual were found to have differing virus strain specificities. Both clones recognized the 11-amino acid peptide RLRDLLLIVTR from position 770-780 of gp41. A change from T to V at position 779 in this epitope abrogated lysis by one clone but not the other. A CTL clone from the other patient, restricted by a different class I HLA allele, recognized the nine-amino acid peptide HRLRDLLLI from position 769-777 of gp41. Of note, the peptide RLRDLLLIVTR has been shown by others to be presented to CTL by HLA-A3.1. Autologous virus sequences from seroconversion and up to 15 wk after presentation in these two patients were recognized by the CTL clones isolated during acute infection. None of the CTL clones recognized the MN strain of HIV-1, indicating the problems inherent in relying on a single virus strain in the development of a vaccine. These studies have identified an immunodominant and promiscuous area for the generation of CTL responses within gp41. This recognition of autologous virus sequences by the initial CTL response is consistent with the hypothesis that a single virus strain is transmitted to the seroconverter and that the CTL response is involved in the initial control of that virus. These studies indicate the importance of the CTL response to HIV-1 infection and have implications in the design of vaccines.

Limiting dilution analysis of cytotoxic T lymphocytes to human immunodeficiency virus gag antigens in infected persons: in vitro quantitation of effector cell populations with p17 and p24 specificities.

The presence of cytotoxic T lymphocytes (CTL) to the gag antigens of human immunodeficiency virus (HIV) has been described in infected populations. We found that the majority of this immune response as measured in bulk CTL assays of unstimulated peripheral blood mononuclear cells (PBMC) is directed against the p24 component of the p55 gag precursor protein. Using limiting dilution analysis of this effector cell population we confirm that the majority of activated gag-specific CTL circulating in the PBMC of infected hemophilic patients are directed at p24 determinants and are present at frequencies of 1/36,000 to 1/86,000 lymphocytes. By performing in vitro stimulation after limiting dilution, the precursor population of gag-specific CTL are characterized and quantitated. HIV gag-specific CTL precursors are identified at frequencies of 1/1700 to 1/17,000 lymphocytes and are made up of cells with both p17 and p24 specificities. No HIV gag-specific CTL precursor cells are identified in the PBMC of HIV-uninfected individuals. These studies demonstrate that CTL directed at both p17 and p24 determinants make up the cellular immune repertoire in HIV-infected individuals but that only the p24-specific CTL are routinely found in an activated state in the circulation.

Cellular targets of infection and route of viral dissemination after an intravaginal inoculation of simian immunodeficiency virus into rhesus macaques.

We used the simian immunodeficiency virus (SIV)/rhesus macaque model to study events that underlie sexual transmission of human immunodeficiency virus type 1 (HIV-1). Four female rhesus macaques were inoculated intravaginally with SIVmac251, and then killed 2, 5, 7, and 9 d later. A technique that detected polymerase chain reaction-amplified SIV in situ showed that the first cellular targets for SIV were in the lamina propria of the cervicovaginal mucosa, immediately subjacent to the epithelium. Phenotypic and localization studies demonstrated that many of the infected cells were likely to be dendritic cells. Within 2 d of inoculation, infected cells were identified in the paracortex and subcapsular sinus of the draining internal iliac lymph nodes. Subsequently, systemic dissemination of SIV was rapid, since culturable virus was detectable in the blood by day 5. From these results, we present a model for mucosal transmission of SIV and HIV-1.

CCR5 levels and expression pattern correlate with infectability by macrophage-tropic HIV-1, in vitro.

Chemokine receptors serve as coreceptors for HIV entry into CD4+ cells. Their expression is thought to determine the tropism of viral strains for different cell types, and also to influence susceptibility to infection and rates of disease progression. Of the chemokine receptors, CCR5 is the most important for viral transmission, since CCR5 is the principal receptor for primary, macrophage-tropic viruses, and individuals homozygous for a defective CCR5 allele (delta32/delta32) are highly resistant to infection with HIV-1. In this study, CCR5-specific mAbs were generated using transfectants expressing high levels of CCR5. The specificity of these mAbs was confirmed using a broad panel of chemokine receptor transfectants, and by their non-reactivity with T cells from delta32/delta32 individuals. CCR5 showed a distinct pattern of expression, being abundant on long-term activated, IL-2-stimulated T cells, on a subset of effector/memory T cells in blood, and on tissue macrophages. A comparison of normal and CCR5 delta32 heterozygotes revealed markedly reduced expression of CCR5 on T cells from the heterozygotes. There was considerable individual to individual variability in the expression of CCR5 on blood T cells, that related to factors other than CCR5 genotype. Low expression of CCR5 correlated with the reduced infectability of T cells with macrophage-tropic HIV-1, in vitro. Anti-CCR5 mAbs inhibited the infection of PBMC by macrophage-tropic HIV-1 in vitro, but did not inhibit infection by T cell-tropic virus. Anti-CCR5 mAbs were poor inhibitors of chemokine binding, indicating that HIV-1 and ligands bind to separate, but overlapping regions of CCR5. These results illustrate many of the important biological features of CCR5, and demonstrate the feasibility of blocking macrophage-tropic HIV-1 entry into cells with an anti-CCR5 reagent.

Functionally inert HIV-specific cytotoxic T lymphocytes do not play a major role in chronically infected adults and children.

The highly sensitive quantitation of virus-specific CD8(+) T cells using major histocompatibility complex-peptide tetramer assays has revealed higher levels of cytotoxic T lymphocytes (CTLs) in acute and chronic virus infections than were recognized previously. However, studies in lymphocytic choriomeningitis virus infection have shown that tetramer assays may include measurement of a substantial number of tetramer-binding cells that are functionally inert. Such phenotypically silent CTLs, which lack cytolytic function and do not produce interferon (IFN)-gamma, have been hypothesized to explain the persistence of virus in the face of a quantitatively large immune response, particularly when CD4 help is impaired. In this study, we examined the role of functionally inert CTLs in chronic HIV infection. Subjects studied included children and adults (n = 42) whose viral loads ranged from <50 to >100,000 RNA copies/ml plasma. Tetramer assays were compared with three functional assays: enzyme-linked immunospot (Elispot), intracellular cytokine staining, and precursor frequency (limiting dilution assay [LDA]) cytotoxicity assays. Strong positive associations were observed between cell numbers derived by the Elispot and the tetramer assay (r = 0.90). An even stronger association between tetramer-derived numbers and intracellular cytokine staining for IFN-gamma was present (r = 0.97). The majority (median 76%) of tetramer-binding cells were consistently detectable via intracellular IFN-gamma cytokine staining. Furthermore, modifications to the LDA, using a low input cell number into each well, enabled LDAs to reach equivalence with the other methods of CTL enumeration. These data together show that functionally inert CTLs do not play a significant role in chronic pediatric or adult HIV infection.

Genotypic and phenotypic characterization of HIV-1 patients with primary infection.

Better characterization of human immunodeficiency virus-type 1 (HIV-1) in patients with primary infection has important implications for the development of an acquired immunodeficiency syndrome (AIDS) vaccine because vaccine strategies should target viral isolates with the properties of transmitted viruses. In five HIV-1 seroconverters, the viral phenotype was found to be uniformly macrophage-tropic and non-syncytium-inducing. Furthermore, the viruses were genotypically homogeneous within each patient, but a common signature sequence was not discernible among transmitted viruses. In the two cases where the sexual partners were also studied, the sequences of the transmitted viruses matched best with minor variants in the blood of the transmitters. There was also a stronger pressure to conserve sequences in gp120 than in gp41, nef, and p17, suggesting that a selective mechanism is involved in transmission.

The immunology of primary HIV infection: which immune responses control HIV replication?

In the majority of patients, HIV-1 replication is quickly and efficiently controlled after the initial burst of viremia. Recent studies have begun to elucidate the underlying immune responses that may be responsible for this dramatic reduction in viral load. These responses include those of HIV-specific CD8+ cytotoxic T lymphocytes and antibodies capable of binding the virus.

Nevirapine synergistically inhibits HIV-1 replication in combination with zidovudine, interferon or CD4 immunoadhesin.

OBJECTIVE: To determine whether synergistic inhibition of HIV-1 replication would result from the in vitro use of nevirapine in combination with zidovudine, interferon (IFN)-alpha 2C, and CD4 immunoadhesin. DESIGN AND METHODS: The non-nucleoside reverse transcriptase inhibitor nevirapine (formerly BI-RG-587) was tested in combination with the other antiretrovial agents. Assays were performed on stimulated peripheral blood lymphocytes infected with HIV-1. Virus replication was assessed by the release of viral p24 antigen into culture supernatants. The median-effect principle was used to assess for synergistic interactions of the combined agents. RESULTS: Zidovudine, IFN-alpha 2C and CD4 immunoadhesin, when used in combination with nevirapine, synergistically inhibited HIV-1 replication in human peripheral blood lymphocytes compared with each agent used alone. Some analyses were also consistent with additive effects, but antagonism was not noted. CONCLUSION: These in vitro findings provide a scientific basis for future trials with similar drug combinations.

Genetic and immunological host factors associated with susceptibility to HIV-1 infection.

The probability of HIV transmission depends on the interplay of many different factors related to infectiousness of the HIV-infected partner, susceptibility of the HIV-uninfected partner, and biological characteristics of HIV strains. Here, we review recent studies of host immunological and genetic factors which may affect susceptibility to HIV-1 infection. These factors are summarized in Table 1. We propose how to explore biological correlates of susceptibility to HIV-1 infection in epidemiological studies, discuss the strengths and limitations of this research, and address the implications for public health.

Antibody-dependent cell-mediated cytotoxicity directed by a human monoclonal antibody reactive with gp120 of HIV-1.

We used a human monoclonal antibody (MAb; 15e) to identify an antibody-dependent cell-mediated cytotoxicity (ADCC) epitope on HIV-1 gp120. 15e has been shown to recognize a conformation-dependent epitope on gp120 which is important in both CD4 binding and neutralizing of HIV-1 infection. 15e binds to gp120 of HIV-1IIIB but not HIV-1RF. Using a standard ADCC assay, 15e was found to mediate ADCC against cells infected with HIV-1IIIB but not HIV-1RF. 15e did not mediate ADCC against cells with recombinant gp120 bound to surface CD4, indicating that 15e does not mediate innocent bystander ADCC against uninfected CD4 cells. To better define the 15e epitope, we performed ADCC against target cells infected with a vaccinia vector which expresses processed HIV-1IIIB gp160 from which the third variable region was deleted (amino acids, 312-328). MAb 15e efficiently mediated ADCC against cells expressing this altered form of gp120, indicating that this region is not contributing to the conformational epitope defined by 15e. 15e defines an important epitope in the human immune response to HIV-1 infection. Antibodies with 15e-like activity may be useful in immunoprophylaxis or immunotherapy of HIV-1 infection.

hu-PBL-SCID mice can be protected from HIV-1 infection by passive transfer of monoclonal antibody to the principal neutralizing determinant of envelope gp120.

OBJECTIVE: To determine whether passive transfer of a monoclonal antibody specific for the principal neutralizing determinant in the V3 region of HIV-1IIIB gp120 can protect mice with severe combined immunodeficiency (SCID) transplanted with normal human peripheral blood leukocytes (hu-PBL), designated hu-PBL-SCID mice, from subsequent challenge with the homologous viral strain. DESIGN AND METHODS: hu-PBL-SCID mice were given intraperitoneal injections of an anti-HIV-1 neutralizing murine monoclonal antibody (BAT123), its mouse-human chimeric form (CGP 47 439), or a control murine antibody (PNTU), at a dose of 40 mg/kg. The mice were then challenged intraperitoneally with 10 mouse infectious doses of HIV-1IIIB. Three weeks later the mice were killed, and spleen cells and peritoneal lavage collected for determination of infection by coculture for viral isolation and by detection of HIV-1 DNA using polymerase chain reaction (PCR). RESULTS: All three antibodies had similar serum half-lives of 9-12 days. No toxicity was observed in the animals. HIV-1 was recovered by coculture from five out of the six mice given PNTU, and by PCR from two out of the six mice given PNTU, but was not recovered by either technique from any of the 12 mice given BAT123 or CGP 47 439. CONCLUSION: BAT123 and CGP 47 439, which are specific for the principal neutralizing determinant of HIV-1IIIB, protect hu-PBL-SCID mice from infection by this viral strain. Our findings support the use of the hu-PBL-SCID mouse as an in vivo model for studying protection against HIV-1 infection by passive immunization with anti-HIV-1 neutralizing antibodies.