Human and simian immunodeficiency viruses (HIV and SIV), influenza virus, and hepatitis C virus (HCV) have heavily glycosylated, highly variable surface proteins. Here we explore N-linked glycosylation site (sequon) variation at the population level in these viruses, using a new Web-based program developed to facilitate the sequon tracking and to define patterns (www.hiv.lanl.gov). This tool allowed rapid visualization of the two distinctive patterns of sequon variation found in HIV-1, HIV-2, and SIV CPZ. The first pattern (fixed) describes readily aligned sites that are either simply present or absent. These sites tend to be occupied by high-mannose glycans. The second pattern (shifting) refers to sites embedded in regions of extreme local length variation and is characterized by shifts in terms of the relative position and local density of sequons; these sites tend to be populated by complex carbohydrates. HIV, with its extreme variation in number and precise location of sequons, does not have a net increase in the number of sites over time at the population level. Primate lentiviral lineages have host species-dependent levels of sequon shifting, with HIV-1 in humans the most extreme. HCV E1 and E2 proteins, despite evolving extremely rapidly through point mutation, show limited sequon variation, although two shifting sites were identified. Human influenza A hemagglutinin H3 HA1 is accumulating sequons over time, but this trend is not evident in any other avian or human influenza A serotypes.

Virus-specific antibodies protect individuals against a wide variety of viral infections. 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.

Summ~aryDuring human immunodeficiency virus (HIV) infection there is a profound and selective decrease in the CD4 + population of T lymphocytes. The mechanism of this depletion is not understood, as only a small fraction of all CD4 + cells appear to be productively infected with HIV-1 in seropositive individuals. In the present study, crosslinking of bound gp120 on human CD4 + T cells followed by signaling through the T cell receptor for antigen was found to result in activation-dependent cell death by a form of cell suicide termed apoptosis, or programmed cell death. The data indicate that even picomolar concentrations of gp120 prime T cells for activationinduced cell death, suggesting a mechanism for CD4 + T cell depletion in acquired immune deficiency syndrome (AIDS), particularly in the face of concurrent infection and antigenic challenge with other organisms. These results also provide an explanation for the enhancement of infection by certain antibodies against HIV, and for the paradox that HIV appears to cause AIDS after the onset of antiviral immunity.T he immunodeficiency that defines AIDS is due primarily to a progressive decline in the number and function of CD4 + T cells. The mechanism of this decline is debated, though lyric infection of cells targeted by interaction of CD4 with the envelope glycoprotein of the HIV virion, gp120, is an obvious model (1-4), and recent data suggest an apoptotic mechanism of cell death after HIV infection (5). However, previous studies have found that only 1 in 1-10 x 104 PBMC actively express HIV-1 in patients with AIDS (6-10), and immune dysfunction is seen early in infection, before a significant proportion of CD4 + cells has been eliminated (11-15). Thus, it is likely that mechanisms other than direct viral destruction contribute to CD4 + T cell loss and to the anergy associated with CD4 + T cell-dependent immune responses.Mouse splenic T cells pretreated with anti-CD4 antibodies die by apoptosis when stimulated through the oL/~ TCR (16). Apoptosis is an active form of physiologic cell death, requiring RNA and protein synthesis, which is characterized by the activation of endogenous endonucleases that cleave chromatin DNA between nucleosomes (17, 18). Here we report that crosslinking of gp120 on human CD4 + T cells followed by signaling through the TCR results in activation-induced cell death. This cell death has the characteristic features of apoptosis, including the histologic changes of nuclear and cytoplasmic condensation and DNA fragmentation into nucleosome-sized multimers of 200 bp. Our data provide a mechanism for the recent observation that CD4 + T cells from HIV-infected individuals are primed in vivo for suicide by apoptosis, upon TCR activation by both superantigen and MHC class II-restricted antigens (19). Materials and Methodslsola~'on ofCD4 + T Cells. Human T ceils were separated from Ficoll-Hypaque-isolated PBMC by rosetting with 2-aminoethylisothio-uronium bromide hydrobromide (AET)-treated SRBC, as described (20). CD4 + calls were isolated by incu...

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The spectrum of human immunodeficiency virus type 1 (HIV-1) isolates neutralized by antibodies from HIV-1-infected humans is broader than the spectrum of isolates neutralized by sera from animals immunized with purified gp120 subunits. This broader neutralization was due, in part, to the presence of antibodies to conserved gp120 conformational epitopes. Purified conformation-dependent gp120-specific human antibodies neutralized a wider range of virus isolates than human antibodies directed to linear determinants in gp120 and were also responsible for the majority of the gp120-specific CD4-blocking activity of HIV-1-infected human sera. A gp120 subunit vaccine that effectively presents these conformation-dependent neutralization epitopes should protect against a broader range of HIV-1 variants than a vaccine that presents exclusively linear determinants.

Prevention of mother to child transmission (MTCT) of HIV remains a major objective where antenatal care is not readily accessible. We tested anti-HIV-1 human neutralizing monoclonal antibodies (NmAb) as post-exposure therapy in an infant macaque model for intrapartum MTCT. One-month-old rhesus macaques were inoculated orally with SHIVSF162P3. On days 1, 4, 7, and 10 after virus exposure, we injected animals subcutaneously with NmAbs and quantified systemic distribution of NmAbs in multiple tissues within 24 h following administration. Replicating virus was found in multiple tissues by day 1 in animals without treatment. All NmAb-treated macaques were free of virus in blood and tissues at 6 months post-exposure. We detected no anti-SHIV T cell responses in blood or tissues at necropsy, and no virus emerged following CD8+ T cell depletion. These results suggest early passive immunotherapy can eliminate early viral foci and thereby prevent the establishment of viral reservoirs.

A 2.4 kb fragment of hCMV (Towne strain), containing the 5' end of the major immediate-early gene, has been cloned, sequenced, and used to construct a series of mammalian cell expression plasmids. The effects of regulatory regions present on this fragment were assessed using human glycoproteins as reporter molecules. We compared secreted levels of Factor VIII, t-PA, and HIV-1 envelope glycoproteins in cells transfected with plasmids in which intron A of the immediate-early gene was present or absent. Secretion of several glycoproteins was significantly higher when cells were transfected with intron A-containing plasmids. Mutation of three basepairs in the strong nuclear factor 1 (NF1) binding site in intron A led to reduced transient expression levels, but not to the level observed in the absence of intron A. Reduced expression from NF1 mutant plasmids was roughly correlated with reduced binding in vitro of NF1 proteins to a synthetic oligonucleotide containing the mutation. The evidence indicates that sequences in intron A positively regulate expression from the hCMV immediate-early enhancer/promoter in transformed monkey kidney cells.

Summary Antibodies have a long history in antiviral therapy, but until recently they have not been actively pursued for HIV-1 due to modest potency and breadth of early human monoclonal antibodies (MAbs) and perceived insurmountable technical, financial, and logistical hurdles. Recent advances in the identification and characterization of MAbs with the ability to potently neutralize diverse HIV-1 isolates has reinvigorated discussion and testing of these products in humans, since new broadly neutralizing MAbs (bnMAbs) are more likely to be effective against worldwide strains of HIV-1. In animal models, there is abundant evidence that bnMAbs can block infection in a dose dependent manner, and the more potent bnMAbs will allow clinical testing at infusion doses that are practically achievable. Moreover, recent advances in antibody engineering are providing further improvements in MAb potency, breadth and half-life. This review summarizes the current state of the field of bnMAb protection in animal models as well as a review of variables that are critical for antiviral activity. Several bnMAbs are currently in clinical testing, and we offer perspectives on their use as pre-exposure prophylaxis (PrEP), potential benefits beyond sterilizing immunity, and a discussion of future approaches to engineer novel molecules.