Ronald Willey

Neutralizing antibody directed against the HIV-1 envelope glycoprotein can completely block HIV-1/SIV chimeric virus infections of macaque monkeys

Virus–specific antibodies protect individuals against a wide variety of viral infections1–7. To assess whether human immunodeficiency virus type 1 (HIV–1) envelope–specific antibodies confer resistance against primate lentivirus infections, we purified immunoglobulin (IgG) from chimpanzees infected with several different HIV–1 isolates, and used this for passive immunization of pig–tailed macaques. These monkeys were subsequently challenged intravenously with a chimeric simian–human immunodeficiency virus (SHIV) bearing an envelope glycoprotein derived form HIV–1DH12, a dual–tropic primary virus isolate. Here we show that anti–SHIV neutralizing activity, determined in vitro using an assay measuring loss of infectivity, is the absolute requirement for antibody–mediated protection in vivo. Using an assay that measures 100% neutralization, the titer in plasma for complete protection of the SHIV–challenged macaques was in the range of 1:5–1:8. The HIV–1–specific neutralizing antibodies studied are able to bind to native gp120 present on infectious virus particles. Administration of non–neutralizing anti–HIV IgG neither inhibited nor enhanced a subsequent SHIV infection.

Mutations within the human immunodeficiency virus type 1 gp160 envelope glycoprotein alter its intracellular transport and processing.

Intracellular transport and processing of the human immunodeficiency virus type 1 (HIV-1) envelope precursor polyprotein, gp160, proceeds via the endoplasmic reticulum (ER) and Golgi complex. We examined gp160 processing during the production of HIV-1 virions in transfected HeLa cells using wild-type and env mutant proviral molecular clones. Results from pulse-chase analyses indicated that a single amino acid substitution within a highly conserved domain of the env gene impaired gp160 export from the ER, leading to an increase in oligomeric forms of gp160 and a decrease in gp120 production. In contrast, gp160 which contained a mutated cleavage site was able to traverse the ER/Golgi complex, even in the absence of proteolytic processing, and become incorporated into budding virions. These findings indicate that export from the ER is a point in the intracellular trafficking of gp160 that is crucial to the production of the mature envelope components.

Generation of the pathogenic R5-tropic simian/human immunodeficiency virus SHIVAD8 by serial passaging in rhesus macaques.

ABSTRACT A new pathogenic R5-tropic simian/human immunodeficiency virus (SHIV) was generated following serial passaging in rhesus macaques. All 13 animals inoculated with SHIVAD8 passaged lineages experienced marked depletions of CD4+ T cells. Ten of these infected monkeys became normal progressors (NPs) and had gradual losses of both memory and naïve CD4+ T lymphocytes, generated antiviral CD4+ and CD8+ T cell responses, and sustained chronic immune activation while maintaining variable levels of plasma viremia (102 to 105 RNA copies/ml for up to 3 years postinfection [p.i.]). To date, five NPs developed AIDS associated with opportunistic infections caused by Pneumocystis carinii, Mycobacterium avium, and Campylobacter coli that required euthanasia between weeks 100 and 199 p.i. Three other NPs have experienced marked depletions of circulating CD4+ T lymphocytes (92 to 154 cells/μl) following 1 to 2 years of infection. When tested for coreceptor usage, the viruses isolated from four NPs at the time of their euthanasia remained R5 tropic. Three of the 13 SHIVAD8-inoculated macaques experienced a rapid-progressor syndrome characterized by sustained plasma viremia of >1 × 107 RNA copies/ml and rapid irreversible loss of memory CD4+ T cells that required euthanasia between weeks 19 and 23 postinfection. The sustained viremia, associated depletion of CD4+ T lymphocytes, and induction of AIDS make the SHIVAD8 lineage of viruses a potentially valuable reagent for vaccine studies.

Control of Viremia and Prevention of Simian-Human Immunodeficiency Virus-Induced Disease in Rhesus Macaques Immunized with Recombinant Vaccinia Viruses plus Inactivated Simian Immunodeficiency Virus and Human Immunodeficiency Virus Type 1 Particles

ABSTRACT An effective vaccine against the human immunodeficiency virus type 1 (HIV-1) will very likely have to elicit both cellular and humoral immune responses to control HIV-1 strains of diverse geographic and genetic origins. We have utilized a pathogenic chimeric simian-human immunodeficiency virus (SHIV) rhesus macaque animal model system to evaluate the protective efficacy of a vaccine regimen that uses recombinant vaccinia viruses expressing simian immunodeficiency virus (SIV) and HIV-1 structural proteins in combination with intact inactivated SIV and HIV-1 particles. Following virus challenge, control animals experienced a rapid and complete loss of CD4+ T cells, sustained high viral loads, and developed clinical disease by 17 to 21 weeks. Although all of the vaccinated monkeys became infected, they displayed reduced postpeak viremia, had no significant loss of CD4+ T cells, and have remained healthy for more than 15 months postinfection. CD8+ T-cell and neutralizing antibody responses in vaccinated animals following challenge were demonstrable. Despite the control of disease, virus was readily isolated from the circulating peripheral blood mononuclear cells of all vaccinees at 22 weeks postchallenge, indicating that immunologic control was incomplete. Virus recovered from the animal with the lowest postchallenge viremia generated high virus loads and an irreversible loss of CD4+ T-cell loss following its inoculation into a naïve animal. These results indicate that despite the protection from SHIV-induced disease, the vaccinated animals still harbored replication-competent and pathogenic virus.

Neutralizing Antibody Titers Conferring Protection to Macaques from a Simian/Human Immunodeficiency Virus Challenge Using the TZM-bl Assay

We previously reported that passive transfer of polyclonal neutralizing antibodies (NAbs) sufficient to generate a titer of 1:38 in the plasma would confer sterilizing protection to 99% of macaques challenged intravenously with 75 TCID(50) of SHIV(DH12). Neutralizing activity in that study was measured in an MT4 cell assay in which infection was completely blocked (EC(100)). In the current study, the TZM-bl system was used to measure EC(50) neutralizing titers in several of the same macaque plasma samples and the relationship between these titers and in vivo protection was determined. The antiviral EC(50) NAb titers measured in individual plasma samples were higher than those previously obtained in the MT4 system. Furthermore, the geometric mean EC(50) NAb titers against pseudotyped SHIV(DH12) were 33-fold greater than the EC(100) titers measured in the MT4 cell assay against the replication-competent SHIV(DH12) inoculated into animals. An augmented probit regression model was used to generate curves relating TZM-bl EC(50) NAb titers and protection from a virus challenge; estimated titers conferring various levels of protection were then determined. In TZM-bl assays using pseudotyped SHIV(DH12), representative percent in vivo protection/estimated EC(50) titers were 99%/1:4467, 90%/1:1175, 80%/1:676, 50%/1:234, and 33%/1:141. Because it is likely that contributions from other arms of the immune system will contribute to vaccine-induced control, the range of EC(50) NAb titers we have derived may be more informative for evaluating the protective value of NAb activity from TZM-bl assays.

Molecular characterization of a polymerase mutant human immunodeficiency virus.

A cell line (8E5) containing a single defective copy of human immunodeficiency virus proviral DNA and producing noninfectious viral particles lacking reverse transcriptase (RT) and endonuclease proteins has recently been described (Folks, et al., (1986b) J. Exp. Med. 164, 280-290). In this report, the mutation in a full-length molecular clone of the provirus (p8E5) was mapped to a 1931-bp region of the pol gene encoding RT. The nucleotide sequence of this segment revealed a 1-base deletion 301 codons from the common amino terminus of the 64- and 51-kDa RTs. Expression of the p8E5 RT segment in Escherichia coli generated an enzymatically inactive and truncated 33-kDa protein.

Kinetics of HIV-1 interactions with sCD4 and CD4+ cells: Implications for inhibition of virus infection and initial steps of virus entry into cells

The mechanisms of human immunodeficiency virus (HIV-1) entry into CD4+ cells and HIV-1 inactivation by sCD4 were studied by analyzing the kinetics of inhibition of viral infection by sCD4 and the kinetics of fusion of CD4+ cells with intact virions labeled with the lipid fluorophore octadecylrhodamine (R18). sCD4 inhibited HIV-1 infection much more effectively when preincubated with virus prior to interaction with CD4+ cells than when mixed simultaneously with virions and cells. The kinetics of inhibition of infection was much slower at 4 degrees and at low sCD4 concentrations than at 37 degrees and at high sCD4 concentrations. In the absence of sCD4, attachment of virus to cells leading to productive infection occurred within 10-30 min. Fusion of the virions with cells started after a 1-2 min lag time and was complete within 15 min. In high-density cell suspensions (5 x 10(7) cells/ml), even very high sCD4 concentrations (100 micrograms/ml) failed to block viral infection during simultaneous mixing of cells, sCD4 and HIV-1. We conclude that the kinetics of sCD4-virus interaction and the competition of sCD4 with the cell surface associated CD4 for the virus are crucial factors in the inhibition of HIV-1 infection by sCD4. These results provide insight into mechanisms of viral penetration into cells and should be considered when designing new approaches for AIDS therapy.

Amino acid deletions are introduced into the V2 region of gp120 during independent pathogenic simian immunodeficiency virus/HIV chimeric virus (SHIV) infections of rhesus monkeys generating variants that are macrophage tropic

Highly pathogenic simian immunodeficiency virus/HIV chimeric viruses (SHIVs) cause extremely rapid, irreversible, and systemic depletions of CD4+ T lymphocytes in inoculated rhesus monkeys. In the absence of this T cell subset, virus production can be sustained for several months by tissue macrophage. During independent infections of seven animals with uncloned virus stocks, SHIV variants emerged bearing amino acid deletions that affected specific residues of the gp120 V2 loop. Some of these macrophage-phase SHIVs replicated to high levels in alveolar macrophage.

Recombination-Mediated Changes in Coreceptor Usage Confer an Augmented Pathogenic Phenotype in a Nonhuman Primate Model of HIV-1-Induced AIDS

ABSTRACT Evolution of the env gene in transmitted R5-tropic human immunodeficiency virus type 1 (HIV-1) strains is the most widely accepted mechanism driving coreceptor switching. In some infected individuals, however, a shift in coreceptor utilization can occur as a result of the reemergence of a cotransmitted, but rapidly controlled, X4 virus. The latter possibility was studied by dually infecting rhesus macaques with X4 and R5 chimeric simian simian/human immunodeficiency viruses (SHIVs) and monitoring the replication status of each virus using specific primer pairs. In one of the infected monkeys, both SHIVs were potently suppressed by week 12 postinoculation, but a burst of viremia at week 51 was accompanied by an unrelenting loss of total CD4+ T cells and the development of clinical disease. PCR analyses of plasma viral RNA indicated an env gene segment containing the V3 region from the inoculated X4 SHIV had been transferred into the genetic background of the input R5 SHIV by intergenomic recombination, creating an X4 virus with novel replicative, serological, and pathogenic properties. These results indicate that the effects of retrovirus recombination in vivo can be functionally profound and may even occur when one of the recombination participants is undetectable in the circulation as cell-free virus.