Malcolm A. Martin

Massive infection and loss of memory CD4 + T cells in multiple tissues during acute SIV infection

It has recently been established that both acute human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections are accompanied by a dramatic and selective loss of memory CD4+ T cells predominantly from the mucosal surfaces. The mechanism underlying this depletion of memory CD4+ T cells (that is, T-helper cells specific to previously encountered pathogens) has not been defined. Using highly sensitive, quantitative polymerase chain reaction together with precise sorting of different subsets of CD4+ T cells in various tissues, we show that this loss is explained by a massive infection of memory CD4+ T cells by the virus. Specifically, 30–60% of CD4+ memory T cells throughout the body are infected by SIV at the peak of infection, and most of these infected cells disappear within four days. Furthermore, our data demonstrate that the depletion of memory CD4+ T cells occurs to a similar extent in all tissues. As a consequence, over one-half of all memory CD4+ T cells in SIV-infected macaques are destroyed directly by viral infection during the acute phase—an insult that certainly heralds subsequent immunodeficiency. Our findings point to the importance of reducing the cell-associated viral load during acute infection through therapeutic or vaccination strategies.

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.

Antibody-mediated immunotherapy of macaques chronically infected with SHIV suppresses viraemia

Neutralizing antibodies can confer immunity to primate lentiviruses by blocking infection in macaque models of AIDS. However, earlier studies of anti-human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies administered to infected individuals or humanized mice reported poor control of virus replication and the rapid emergence of resistant variants. A new generation of anti-HIV-1 monoclonal antibodies, possessing extraordinary potency and breadth of neutralizing activity, has recently been isolated from infected individuals. These neutralizing antibodies target different regions of the HIV-1 envelope glycoprotein including the CD4-binding site, glycans located in the V1/V2, V3 and V4 regions, and the membrane proximal external region of gp41 (refs 9, 10, 11, 12, 13, 14). Here we have examined two of the new antibodies, directed to the CD4-binding site and the V3 region (3BNC117 and 10-1074, respectively), for their ability to block infection and suppress viraemia in macaques infected with the R5 tropic simian–human immunodeficiency virus (SHIV)-AD8, which emulates many of the pathogenic and immunogenic properties of HIV-1 during infections of rhesus macaques. Either antibody alone can potently block virus acquisition. When administered individually to recently infected macaques, the 10-1074 antibody caused a rapid decline in virus load to undetectable levels for 4–7 days, followed by virus rebound during which neutralization-resistant variants became detectable. When administered together, a single treatment rapidly suppressed plasma viraemia for 3–5 weeks in some long-term chronically SHIV-infected animals with low CD4+ T-cell levels. A second cycle of anti-HIV-1 monoclonal antibody therapy, administered to two previously treated animals, successfully controlled virus rebound. These results indicate that immunotherapy or a combination of immunotherapy plus conventional antiretroviral drugs might be useful as a treatment for chronically HIV-1-infected individuals experiencing immune dysfunction.

HIV vaccine research: the way forward.

The need to broaden research directed at answering fundamental questions in HIV vaccine discovery through laboratory, nonhuman primate (NHP), and clinical research has recently been emphasized. In addition, the importance of attracting and retaining young researchers, developing better NHP models, and more closely linking NHP and clinical research is being stressed. In an era of a level budget for biomedical research at the U.S. National Institutes of Health (NIH), HIV/AIDS vaccine research efforts will need to be carefully prioritized such that resources to energize HIV vaccine discovery can be identified. This article summarizes progress and challenges in HIV vaccine research, the priorities arising from a recent summit at NIAID, and the actions needed, some already under way, to address those priorities.

The role of human immunodeficiency virus type 1 envelope glycoproteins in virus infection.

Enveloped viruses enter cells by a two-step process. The first step involves the binding of a viral surface protein to receptors on the plasma membrane of the host cell. After receptor binding, a membrane fusion reaction takes place between the lipid bilayer of the viral envelope and host cell membranes. This fusion reaction can occur either at the plasma membrane or in acidic endosomes following receptor-mediated endocytosis. In either case, the membrane fusion reaction delivers the viral nucleocapsid into the host cytoplasm, allowing the infection to proceed. Viral proteins embedded in the lipid bilayer of the viral envelope (known variously as surface, spike, or envelope proteins) catalyze receptor binding and membrane fusion reactions. The critical involvement of these viral proteins in receptor binding and membrane fusion has stimulated intensive investigation aimed at understanding the mechanisms by which these proteins function. In this article, we provide a brief overview of the roles envelope (Env) glycoproteins play in the human immunodeficiency virus type 1 (HIV-1) life cycle.

Quantitation of simian virus 40 sequences in African green monkey, mouse and virus-transformed cell genomes

The number of SV40‡ genome equivalents present in green monkey and SV40 transformed mammalian DNAs have been evaluated by measuring DNA reassociation kinetics on hydroxyapatite. Under the proper conditions, this method is sufficiently sensitive to detect less than one SV40 DNA molecule per mammalian genome (one part in 106) as shown by reconstruction experiments. In four out of five SV40 transformed lines examined, an average of one SV40 genome equivalent was present in the cell DNA; three SV40 DNA equivalents per cell were found in the fifth viral transformed line. The background level of SV40 DNA sequences within the 3T3 genome was 0.45 equivalent per cell. An average of 0.5 SV40 genome equivalent was measured per African green monkey genome, an amount too small to be reliably detected using DNA-DNA hybridization on nitrocellulose membranes. The biological significance of these results and their relationship to previously reported values are discussed.

Infection and Pathogenicity of Chimeric Simian-Human Immunodeficiency Viruses in Macaques: Determinants of High Virus Loads and CD4 Cell Killing

Chimeric simian-human immunodeficiency viruses (SHIVs) carrying envelope glycoproteins derived from a T cell-macrophage dual-tropic primary isolate (human immunodeficiency virus type 1 [HIV-1] strain DH12) were constructed. When inoculated into macaque monkeys, SHIV(MD14) carrying simian immunodeficiency virus-derived nef established significantly higher virus loads than did SHIV(MD1), which contains the HIV-1 nef gene. Three patterns of CD4 cell depletion were observed in infected monkeys: exponential and irreversible loss to undetectable levels within 10 weeks of infection; marked reduction during acute infection followed by partial recovery and stabilization (lasting from 10 weeks to > 1 year), with a later decline to undetectable levels in some animals; and a transient loss during acute infection. The induced immunodeficiency was accompanied by CD4 cell counts of < 50 cells/microL and was associated with Pneumocystis carinii pneumonia, cytomegalovirus meningoencephalitis, lymphoid depletion, and thymic atrophy.

Passive transfer of modest titers of potent and broadly neutralizing anti-HIV monoclonal antibodies block SHIV infection in macaques

Five potent and broadly anti-HIV neutralizing monoclonal antibodies are able to block infection by two different SHIVs in monkeys. The authors show that antibodies targeting the outer glycan coat were the most effective and determined that titers of roughly 1:100 protected half the animals.

Human immunodeficiency virus type 1 neutralizing antibodies accelerate clearance of cell-free virions from blood plasma

The concentration of human immunodeficiency virus type 1 (HIV–1) particles in blood plasma is very predictive of the subsequent disease course in an infected individual; its measurement has become one of the most important parameters for monitoring clinical status. Steady–state virus levels in plasma reflect a balance between the rates of virions entering and leaving the peripheral blood. We analyzed the rate of virus clearance in the general circulation in rhesus macaques receiving a continuous infusion of cell–free particles in the presence and absence of virus–specific antibodies. Here we show, by measuring virion RNA, particle–associated p24 Gag protein and virus infectivity, that the clearance of physical and infectious particles from a primary, dual–tropic virus isolate, HIV–1DH12, is very rapid in naive animals, with half–lives ranging from 13 to 26 minutes. In the presence of high–titer HIV–1DH12–specific neutralizing antibodies, the half–life of virion RNA was considerably reduced (to 3.9–7.2 minutes), and infectious virus in the blood became undetectable. Although physical virus particles were eliminated extravascularly, the loss of virus infectivity in the blood reflected the combined effects of extravascular clearance and intravascular inactivation of HIV–1 infectivity due to antibody binding.

N-Linked Glycosylation Sites Adjacent to and within the V1/V2 and the V3 Loops of Dualtropic Human Immunodeficiency Virus Type 1 Isolate DH12 gp120 Affect Coreceptor Usage and Cellular Tropism

ABSTRACT The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) is extensively glycosylated, containing approximately 23 asparagine (N)-linked glycosylation sites on its gp120 subunit. In this study, specific glycosylation sites on gp120 of a dualtropic primary HIV-1 isolate, DH12, were eliminated by site-directed mutagenesis and the properties of the resulting mutant envelopes were evaluated using a recombinant vaccinia virus-based cell-to-cell fusion assay alone or in the context of viral infections. Of the glycosylation sites that were evaluated, those proximal to the V1/V2 loops (N135, N141, N156, N160) and the V3 loops (N301) of gp120 were functionally critical. The glycosylation site mutations near the V1/V2 loop compromised the use of CCR5 and CXCR4 equally. In contrast, a mutation within the V3 loop preferentially inhibited the usage of CCR5; although this mutant protein completely lost its CCR5-dependent fusion activity, it retained 50% of the wild-type fusion activity with CXCR4. The replication of a virus containing this mutation was severely compromised in peripheral blood mononuclear cells, MT-4 cells, and primary monocyte-derived macrophages. A revertant virus, which acquired second site changes in the V3 loop that resulted in an increase in net positive charge, was isolated. The revertant virus fully recovered the usage of CXCR4 but not of CCR5, thereby altering the tropism of the parental virus from dualtropic to T-tropic. These results suggest that carbohydrate moieties near the V1/V2 and the V3 loops play critical roles in maintaining proper conformation of the variable loops for optimal interaction with receptors. Our results, combined with those of previously reported studies, further demonstrate that the function of individual glycans may be virus isolate dependent.