Nucleic Acid, Antibody, and Virus Culture Methods to Detect Xenotropic MLV-Related Virus in Human Blood Samples.

The MLV-related retrovirus, XMRV, was recently identified and reported to be associated with both prostate cancer and chronic fatigue syndrome. At the National Cancer Institute-Frederick, MD (NCI-Frederick), we developed highly sensitive methods to detect XMRV nucleic acids, antibodies, and replication competent virus. Analysis of XMRV-spiked samples and/or specimens from two pigtail macaques experimentally inoculated with 22Rv1 cell-derived XMRV confirmed the ability of the assays used to detect XMRV RNA and DNA, and culture isolatable virus when present, along with XMRV reactive antibody responses. Using these assays, we did not detect evidence of XMRV in blood samples (N = 134) or prostate specimens (N = 19) from two independent cohorts of patients with prostate cancer. Previous studies detected XMRV in prostate tissues. In the present study, we primarily investigated the levels of XMRV in blood plasma samples collected from patients with prostate cancer. These results demonstrate that while XMRV-related assays developed at the NCI-Frederick can readily measure XMRV nucleic acids, antibodies, and replication competent virus, no evidence of XMRV was found in the blood of patients with prostate cancer.

Compromised gastrointestinal integrity in pigtail macaques is associated with increased microbial translocation, immune activation, and IL-17 production in the absence of SIV infection.

Pigtail macaques (PTMs) rapidly progress to AIDS after simian immunodeficiency virus (SIV) infection. Given the strong association between human immunodeficiency virus (HIV) and SIV disease progression and microbial translocation and immune activation, we assessed whether high basal levels of immune activation and microbial translocation exist in PTMs. We found that before SIV infection, PTMs had high levels of microbial translocation that correlated with significant damage to the structural barrier of the gastrointestinal tract. Moreover, this increased microbial translocation correlated with high levels of immune activation and was associated with high frequencies of interleukin-17-producing T cells. These data highlight the relationship among mucosal damage, microbial translocation and systemic immune activation in the absence of SIV replication, and underscore the importance of microbial translocation in the rapid course of disease progression in SIV-infected PTMs. Furthermore, these data suggest that PTM may be an ideal model to study therapeutic interventions aimed at decreasing microbial translocation-induced immune activation.

A fusion inhibitor prevents spread of immunodeficiency viruses, but not activation of virus-specific T cells, by dendritic cells.

Dendritic cells (DCs) play a key role in innate immune responses, and their interactions with T cells are critical for the induction of adaptive immunity. However, immunodeficiency viruses are efficiently captured by DCs and can be transmitted to and amplified in CD4(+) T cells, with potentially deleterious effects on the induction of immune responses. In DC-T-cell cocultures, contact with CD4(+), not CD8(+), T cells preferentially facilitated virus movement to and release at immature and mature DC-T-cell contact sites. This occurred within 5 min of DC-T-cell contact. While the fusion inhibitor T-1249 did not prevent virus capture by DCs or the release of viruses at the DC-T-cell contact points, it readily blocked virus transfer to and amplification in CD4(+) T cells. Higher doses of T-1249 were needed to block the more robust replication driven by mature DCs. Virus accumulated in DCs within T-1249-treated cocultures but these DCs were actually less infectious than DCs isolated from untreated cocultures. Importantly, T-1249 did not interfere with the stimulation of virus-specific CD4(+) and CD8(+) T-cell responses when present during virus-loading of DCs or for the time of the DC-T-cell coculture. These results provide clues to identifying strategies to prevent DC-driven virus amplification in CD4(+) T cells while maintaining virus-specific immunity, an objective critical in the development of microbicides and therapeutic vaccines.

Evaluation of aldrithiol-2-inactivated preparations of HIV type 1 subtypes A, B, and D as reagents to monitor T cell responses.

The development of HIV vaccines is an urgent priority and there is need to generate reagents representing multiple subtypes that can be used to screen HIV-1-specific responses. We used Aldrithiol-2 (AT-2), a mild oxidizing reagent, to eliminate the infectivity of HIV while maintaining its structure and ability to be processed for presentation to T cells. Inactivated subtype A, B, and D viruses were evaluated for their ability to stimulate T cell responses in PBMC samples from 18 U.S. subjects infected with HIV-1 subtype B and 32 Ugandan subjects infected with subtypes A and D or recombinants AC and AD. Five HIV-1-negative samples were also analyzed. T cell responses to AT-2-inactivated viral isolates were monitored by interferon-gamma (IFN-gamma) intracellular cytokine secretion (ICS) analysis; matched microvesicle preparations served as negative controls. Among the 18 subtype B infected subjects, 39% had CD3(+) CD4 (+) IFN-gamma responses and 67% had CD3(+) CD8(+) IFN-gamma responses. Of the 32 Ugandan subjects, 34% demonstrated CD3(+) CD4(+) IFN-gamma responses and 78% demonstrated CD3(+) CD8(+) IFN-gamma responses. Both subtype-specific and cross-reactive responses were observed. Responses to the AT-2 viruses tended to be lower in magnitude than those detected by a set of overlapping gag peptides. Robust lymphoproliferative responses to AT-2 viruses were seen in a subset of subjects. In conclusion, AT-2-inactivated HIV-1 virions stimulated both CD4 and CD8 HIV-1-specific responses and may provide an additional reagent for screening HIV-1-specific responses in HIV seropositives and vaccinees.

CpG-C ISS-ODN activation of blood-derived B cells from healthy and chronic immunodeficiency virus-infected macaques.

Cytosine-phosphate-guanine class C (CpG-C) immunostimulatory sequence oligodeoxynucleotides (ISS-ODNs) activate human B cells and dendritic cells (DCs), properties that suggest potential use as a novel adjuvant to enhance vaccine efficacy. After demonstrating that the CpG-C ISS-ODN C274 activates macaque DCs, we examined in vitro activation of macaque B cells by C274 as a prelude to evaluation of this molecule as an adjuvant in the testing of candidate human immunodeficiency virus vaccines in the rhesus macaque-simian immunodeficiency virus (SIV) model. C274 induced macaque CD20(+) B cells to proliferate more strongly than CD40 ligand or CpG-B ISS-ODN. C274 enhanced B cell survival; increased viability was most evident after 3-7 days of culture. Increased expression of CD40, CD80, and CD86 by B cells was apparent within 24 h of exposure to C274 and persisted for up to 1 week. C274-stimulated, B cell-enriched and peripheral blood mononuclear cell suspensions from naïve and immunodeficiency virus-infected monkeys secreted several cytokines [e.g., interleukin (IL)-3, IL-6, IL-12, interferon-alpha] and chemokines [e.g., monocyte chemoattractant protein-1/CC chemokine ligand 2 (CCL2), macrophage-inflammatory protein-1alpha/CCL3, IL-8/CXC chemokine ligand 8]. In comparison, exposure of macaque B cells to SIV had minimal impact on surface phenotype, despite inducing cytokine and chemokine production in cells from infected and uninfected animals. These observations emphasize the need to identify strategies to optimally boost immune function, as immunodeficiency viruses themselves only partially activate B cells and DCs. The ability of C274 to stimulate B cells and DCs in healthy and infected monkeys suggests its possible use as a broad-acting adjuvant to be applied in the rhesus macaque model for the development of preventative and therapeutic vaccines.

Macaque dendritic cells infected with SIV-recombinant canarypox ex vivo induce SIV-specific immune responses in vivo.

Dendritic cells (DCs) infected with recombinant avipox vectors express the introduced genes and activate antigen-specific T cells. DCs exhibit distinct differentiation-dependent immune functions. Moreover, immature DCs are readily infected by canarypox vectors, but undergo tumor necrosis factor (TNF)-alpha-dependent death, while fewer mature DCs get infected and resist dying. A pilot study was performed using the rhesus macaque system to explore whether immature and mature DCs infected with SIV-recombinant canarypox (vCP180) ex vivo could induce primary virus-specific immune responses in vivo. After subcutaneous (sc) reinjection, functional monocyte-derived DCs migrated to lymph nodes (LNs) within 1-2 days and primed T cells in vivo. This was observed by monitoring dye-labeled DCs in the draining LNs and tetanus toxoid (TT)-specific T cell responses after injection of TT-loaded DCs. DCs from simian immunodeficiency virus (SIV)-naïve rhesus macaques were infected with vCP180 (SIVmac142 gag, pol, and env genes), and sc reinjected into donor animals. Low-level SIV-specific T cell proliferation, but little if any interferon (IFN)-gamma production was detected. DCs pulsed with vCP180 in combination with TT and keyhole limpet hemocyanin (KLH) (to activate additional T cells and provide "helper" cytokines) induced SIV-, TT-, and KLH-specific T cell responses, including IFN-gamma responses not seen when vCP180-carrying DCs were used alone. Interleukin (IL)-10 and low-level antibody responses were also observed. This pilot study provides the proof of principle that sc injected ex vivo SIV-recombinant canarypox-infected DCs safely induce low-level SIV-specific immune responses in vivo.

Transient early post-inoculation anti-retroviral treatment facilitates controlled infection with sparing of CD4+ T cells in gut-associated lymphoid tissues in SIVmac239-infected rhesus macaques, but not resistance to rechallenge.

Like human immunodeficiency virus infection of humans, infection of rhesus macaques with pathogenic simian immunodeficiency virus (SIV) strains typically results in persistent progressive infection, leading to clinically significant immunosuppression. In previous studies, we administered short term anti-retroviral treatment, shortly after intravenous inoculation with SIVsmE660, in an effort to allow immunologic sensitization under conditions not characterized by overwhelming cytopathic infection compromising the developing immune response. We showed that such treatment allowed control of off treatment viremia and was associated with resistance to rechallenge. Control of off treatment viremia was associated, at least in part, with CD8+ lymphocytes, based on in vivo CD8 depletion studies. In the present study, six rhesus macaques were infected intravenously with 100 MID50 of SIVmac239; four then received 30 days of treatment with tenofovir 9-[2-(R)-(phosphonomethoxy)propyl]adenine (PMPA); 20-30 mg/kg, subcutaneously) starting 24 hours post-inoculation. Tenofovir-treated animals showed low (<500 copy Eq/ml) or undetectable (<100 copy Eq/ml) plasma SIV RNA levels during treatment, with undetectable plasma viremia following discontinuation of treatment. Plasma SIV RNA remained <100 copy Eq/ml, even after depletion of CD8+ lymphocytes, 6 weeks after discontinuation of tenofovir treatment. In contrast to untreated infected control animals that showed substantial depletion of CD4+ T cells from gut-associated lymphoid tissues (GALT), tenofovir-treated animals showed sparing of GALT CD4+ T cells both during the treatment period and in the off treatment follow-up period. However, in contrast to earlier results with animals infected with SIVsmE660, in the present study, the animals did not develop readily measurable cellular anti-SIV immune responses, and did not resist homologous rechallenge with SIVmac239, administered 44 weeks after the initial infection. Differences in the animals and virus strains employed may in part account for the differences in results observed. Comparative analysis of virologic and immunologic parameters in this model system may provide important insights for understanding the basis of effective immunologic control of SIV infection.

Longitudinal follow up of SIVmac pathogenesis in rhesus macaques of Chinese origin: emergence of B cell lymphoma.

Two subspecies of rhesus (Rh) macaques, the Chinese (Ch) and Indian (Ind) subspecies were infected intravenously with 100TCID50 SIVmac239. CD4+, CD8+ T cells, plasma viral loads, depletion of intestinal lymphocytes with memory phenotype, humoral immune responses and clinical courses were monitored for 600 days. The pathogenesis of SIVmac was also compared with primary human immunodeficiency virus (HIV) infection of humans. Plasma viral loads in Ch Rh were lower in the acute and chronic phases compared with Ind Rh. SIVmac pathogenesis in Ch Rh was closer to virus loads in untreated HIV infected humans. Ch Rh had higher CD4/CD8 ratios, stronger antibody responses and interestingly, less depletion of intestinal memory CCR5+ CD4+ T lymphocytes compared with Ind Rh. One Ch Rh developed B cell origin lymphoma at 570 days post-infection, the first such report in this subspecies. Three of four Ind Rh developed AIDS within 6 months. The findings indicate that Ch Rh are more resistant to SIVmac pathogenesis compared with Ind Rh and that Ch Rh paralleled HIV-1 infections in untreated adult humans. The SIVmac infected Ch Rh subspecies are an acceptable model for HIV/AIDS.

Whole inactivated SIV virion vaccines with functional envelope glycoproteins: safety, immunogenicity, and activity against intrarectal challenge.

A novel type of whole inactivated simian immunodeficiency virus (SIV) virion vaccine immunogen with functional envelope glycoproteins was evaluated, without adjuvant, in rhesus macaques. Immunogens included purified inactivated virions of SIVmac239, a designed mutant of SIVmac239 with gp120 carbohydrate attachment sites deleted (SIVmac239 g4,5), and SIVmneE11S. The vaccines were noninfectious, safe, and immunogenic, inducing antibody responses and cellular responses, including responses by CD8+ lymphocytes. Interpretation of protective efficacy following intrarectal challenge was complicated by incomplete take of the challenge in some SIV naïve controls.

Infectious and whole inactivated simian immunodeficiency viruses interact similarly with primate dendritic cells (DCs): differential intracellular fate of virions in mature and immature DCs.

As potential targets for human immunodeficiency virus type 1 and simian immunodeficiency virus (HIV-1 and SIV), dendritic cells (DCs) likely play a significant role in the onset and spread of infection as well as in the induction of antiviral immunity. Using the SIV-macaque system to study the very early events in DC-virus interactions, we compared chemically inactivated SIV having conformationally and functionally intact envelope glycoproteins (2,2'-dithiodipyridine [AT-2] SIV) to infectious and heat-treated SIV. Both human and macaque DCs interact similarly with SIV without detectable effects on DC viability, phenotype, or endocytic function. As assessed by measuring cell-associated viral RNA, considerable amounts of virus are captured by the DCs and this is reduced when the virus is heat treated or derived from a strain that expresses low levels of envelope glycoprotein. Immunostaining for SIV proteins and electron microscopy indicated that few intact virus particles are retained at the periphery of the endocytically active, immature DCs. This contrasts with a perinuclear localization of numerous virions in large vesicular compartments deeper within mature DCs (in which macropinocytosis is down-regulated). Both immature and mature DCs are capable of clathrin-coated pit-mediated uptake of SIV, supporting the notion that the receptor-mediated uptake of virus can occur readily in mature DCs. While large numbers of whole viruses were preferentially found in mature DCs, both immature and mature DCs contained similar amounts of viral RNA, suggesting that different uptake/virus entry mechanisms are active in immature and mature DCs. These findings have significant implications for cell-to-cell transmission of HIV-1 and SIV and support the use of AT-2 SIV, an authentic but noninfectious form of virus, as a useful tool for studies of processing and presentation of AT-2 SIV antigens by DCs.

Role of CD8(+) lymphocytes in control of simian immunodeficiency virus infection and resistance to rechallenge after transient early antiretroviral treatment.

Transient antiretroviral treatment with tenofovir, (R)-9-(2-phosphonylmethoxypropyl)adenine, begun shortly after inoculation of rhesus macaques with the highly pathogenic simian immunodeficiency virus (SIV) isolate SIVsmE660, facilitated the development of SIV-specific lymphoproliferative responses and sustained effective control of the infection following drug discontinuation. Animals that controlled plasma viremia following transient postinoculation treatment showed substantial resistance to subsequent intravenous rechallenge with homologous (SIVsmE660) and highly heterologous (SIVmac239) SIV isolates, up to more than 1 year later, despite the absence of measurable neutralizing antibody. In some instances, resistance to rechallenge was observed despite the absence of detectable SIV-specific binding antibody and in the face of SIV lymphoproliferative responses that were low or undetectable at the time of challenge. In vivo monoclonal antibody depletion experiments demonstrated a critical role for CD8(+) lymphocytes in the control of viral replication; plasma viremia rose by as much as five log units after depletion of CD8(+) cells and returned to predepletion levels (as low as <100 copy Eq/ml) as circulating CD8(+) cells were restored. The extent of host control of replication of highly pathogenic SIV strains and the level of resistance to heterologous rechallenge achieved following transient postinoculation treatment compared favorably to the results seen after SIVsmE660 and SIVmac239 challenge with many vaccine strategies. This impressive control of viral replication was observed despite comparatively modest measured immune responses, less than those often achieved with vaccination regimens. The results help establish the underlying feasibility of efforts to develop vaccines for the prevention of AIDS, although the exact nature of the protective host responses involved remains to be elucidated.

Emergence and kinetics of simian immunodeficiency virus-specific CD8(+) T cells in the intestines of macaques during primary infection.

In this report, three Mamu-A*01(+) rhesus macaques were examined to compare the emergence of simian immunodeficiency virus (SIV)-specific CD8(+) T cells in the intestines and blood in early SIV infection using a major histocompatibility complex class I tetramer complexed with the Gag(181-189) peptide. Fourteen days after intravenous inoculation with SIVmac251, large numbers of SIV Gag(181-189)-specific CD8(+) T cells were detected in the intestinal mucosa (3.1 to 11.5% of CD3(+) CD8(+) lymphocytes) as well as in the blood (3.1 to 13.4%) of all three macaques. By 21 days postinoculation, levels of tetramer-binding cells had dropped in both the intestines and blood. At day 63, however, levels of SIV Gag(181-189)-specific CD8(+) T cells in the intestines had rebounded in all three macaques to levels that were higher (8.6 to 18.7%) than those at day 21. In contrast, percentages of tetramer-binding cells in the peripheral blood remained comparatively stable (2.5 to 4.5%) at this time point. In summary, SIV Gag(181-189)-specific CD8(+) T cells appeared in both the intestinal mucosa and peripheral blood at a comparable rate and magnitude in primary SIV infection. Given that the intestine is a major site of early viral replication as well as the site where most of the total body lymphocyte pool resides, these data indicate that it is also an early and important site of development of antiviral immune responses.

Sequential immunization of macaques with two differentially attenuated vaccines induced long-term virus-specific immune responses and conferred protection against AIDS caused by heterologous simian human immunodeficiency Virus (SHIV(89.6)P).

Four rhesus macaques were sequentially immunized with live vaccines DeltavpuDeltanefSHIV-4 (vaccine-I) and Deltavpu SHIV(PPC) (vaccine-II). The vaccine viruses did not replicate productively in the peripheral blood mononuclear cells (PBMCs) of the vaccinated animals. All four animals developed binding antibodies against both the vaccine-I and -II envelope glycoproteins but neutralizing antibodies only against vaccine-I. They developed vaccine virus-specific CTLs that also recognized homologous as well as heterologous pathogenic SHIVs. Thirty weeks after the last immunization, the vaccinated animals and three unvaccinated control animals were challenged iv with a highly virulent heterologous SHIV(89.6)P. As expected, the three unvaccinated control animals developed large numbers of infectious PBMCs, high plasma viremia, and precipitous loss of CD4(+) T cells. Two controls did not develop any immune response and succumbed to AIDS in about 6 months. The third control animal developed neutralizing antibodies and had a more chronic disease course, but eventually succumbed to AIDS-related complications 81 weeks after inoculation. The four vaccinated animals became infected with challenge virus as indicated by the presence of challenge virus-specific DNA in the PBMCs and RNA in plasma. However, virus in these animals replicated approximately 200- to 60,000-fold less efficiently than in control animals and eventually, plasma viral RNA became undetectable in three of the four vaccinates. The animals maintained normal CD4(+) T-cell levels throughout the observation period of 85 weeks after a transient drop at Week 3 postchallenge. They also maintained CTL responses throughout the observation period. These studies thus showed that the graded immunization schedule resulted in a safe and highly effective long-lasting immune response that was associated with protection against AIDS by highly pathogenic heterologous SHIV(89.6)P.

Immunogenicity and protective efficacy of oligomeric human immunodeficiency virus type 1 gp140.

The biologically active form of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein is oligomeric. We previously described a soluble HIV-1 IIIB Env protein, gp140, with a stable oligomeric structure composed of uncleaved gp120 linked to the ectodomain of gp41 (P. L. Earl, C. C. Broder, D. Long, S. A. Lee, J. Peterson, S. Chakrabarti, R. W. Doms, and B. Moss, J. Virol. 68:3015-3026, 1994). Here we compared the antibody responses of rabbits to gp120 and gp140 that had been produced and purified in an identical manner. The gp140 antisera exhibited enhanced cross-reactivity with heterologous Env proteins as well as greater neutralization of HIV-1 compared to the gp120 antisera. To examine both immunogenicity and protective efficacy, we immunized rhesus macaques with oligomeric gp140. Strong neutralizing antibodies against a homologous virus and modest neutralization of heterologous laboratory-adapted isolates were elicited. No neutralization of primary isolates was observed. However, a substantial fraction of the neutralizing activity could not be blocked by a V3 loop peptide. After intravenous challenge with simian-HIV virus SHIV-HXB2, three of the four vaccinated macaques exhibited no evidence of virus replication.

In vivo attenuation of simian immunodeficiency virus by disruption of a tyrosine-dependent sorting signal in the envelope glycoprotein cytoplasmic tail.

Attenuated simian immunodeficiency viruses (SIVs) have been described that produce low levels of plasma virion RNA and exhibit a reduced capacity to cause disease. These viruses are particularly useful in identifying viral determinants of pathogenesis. In the present study, we show that mutation of a highly conserved tyrosine (Tyr)-containing motif (Yxxphi) in the envelope glycoprotein (Env) cytoplasmic tail (amino acids YRPV at positions 721 to 724) can profoundly reduce the in vivo pathogenicity of SIVmac239. This domain constitutes both a potent endocytosis signal that reduces Env expression on infected cells and a sorting signal that directs Env expression to the basolateral surface of polarized cells. Rhesus macaques were inoculated with SIVmac239 control or SIVmac239 containing either a Tyr-721-to-Ile mutation (SIVmac239Y/I) or a deletion of Tyr-721 and the preceding glycine (DeltaGY). To assess the in vivo replication competence, all viruses contained a stop codon in nef that has been shown to revert during in vivo but not in vitro replication. All three control animals developed high viral loads and disease. One of two animals that received SIVmac239Y/I and two of three animals that received SIVmac239DeltaGY remained healthy for up to 140 weeks with low to undetectable plasma viral RNA levels and normal CD4(+) T-cell percentages. These animals exhibited ongoing viral replication as determined by detection of viral sequences and culturing of mutant viruses from peripheral blood mononuclear cells and persistent anti-SIV antibody titers. In one animal that received SIVmac239Y/I, the Ile reverted to a Tyr and was associated with a high plasma RNA level and disease, while one animal that received SIVmac239DeltaGY also developed a high viral load that was associated with novel and possibly compensatory mutations in the TM cytoplasmic domain. In all control and experimental animals, the nef stop codon reverted to an open reading frame within the first 2 months of inoculation, indicating that the mutant viruses had replicated well enough to repair this mutation. These findings indicate that the Yxxphi signal plays an important role in SIV pathogenesis. Moreover, because mutations in this motif may attenuate SIV through mechanisms that are distinct from those caused by mutations in nef, this Tyr-based sorting signal represents a novel target for future models of SIV and human immunodeficiency virus attenuation that could be useful in new vaccine strategies.

Mucosal challenge of Macaca nemestrina with simian immunodeficiency virus (SIV) following SIV nucleocapsid mutant DNA vaccination.

A simian immunodeficiency virus (SIV)(Mne) DNA clone was constructed that produces viruses containing a four amino acid deletion in the second zinc finger of the nucleocapsid (NC) domain of the Gag polyprotein. Viruses produced from this clone, although non-infectious both in vitro and in vivo, complete a majority of the steps in a single retroviral infection cycle. Eight pig-tailed macaques (Macaca nemestrina) were inoculated intramuscularly and subcutaneously three times over the course of 24 weeks with the NC mutant expressing DNA. These macaques, and four controls, were then challenged mucosally (intrarectally) with the homologous virus (SIV Mne CL E11S) and monitored for evidence of infection and clinical disease. Prior to challenge, a measurable humoral immune response was noted in four of eight immunized macaques. After challenge, all 12 macaques became infected, although four immunized animals greatly restricted their viral replication, and one immunized animal that controlled replication remains antibody negative. No disease has been evidence during the 46-week period of monitoring after challenge.

Vaccine protection against simian immunodeficiency virus by recombinant strains of herpes simplex virus.

An effective vaccine for AIDS may require development of novel vectors capable of eliciting long-lasting immune responses. Here we report the development and use of replication-competent and replication-defective strains of recombinant herpes simplex virus (HSV) that express envelope and Nef antigens of simian immunodeficiency virus (SIV). The HSV recombinants induced antienvelope antibody responses that persisted at relatively stable levels for months after the last administration. Two of seven rhesus monkeys vaccinated with recombinant HSV were solidly protected, and another showed a sustained reduction in viral load following rectal challenge with pathogenic SIVmac239 at 22 weeks following the last vaccine administration. HSV vectors thus show great promise for being able to elicit persistent immune responses and to provide durable protection against AIDS.

Real-time polymerase chain reaction assay for cell-associated HTLV type I DNA viral load.

We have developed a quantitative real-time PCR assay for HTLV-I DNA. This assay approach uses real-time monitoring of fluorescent signal generation as a consequence of Taq-mediated amplification of specific target sequences to allow real-time kinetic analysis of amplicon production. This kinetic approach yields excellent sensitivity and an extremely broad linear dynamic range, and ensures that quantitation is based on analysis during the exponential phase of amplification, regardless of the input template copy number. The HTLV-I DNA assay has a nominal threshold sensitivity of 10 copy Eq/reaction, although single-copy plasmid template can be detected at frequencies consistent with statistical prediction. The linear dynamic range is in excess of 5 logs. Interassay reproducibility averages 14% (coefficient of variation) for control templates over a range of 10(1) to 10(6) copy Eq/reaction and 25%, based on studies of extraction and analysis of replicate aliquots of PBMC specimens from HTLV-I-infected subjects. The primer/probe combination targets tax sequences conserved across described HTLV-I and HTLV-II isolates. Parallel quantitation in the same samples of an endogenous sequence present at a known copy number per cell allows normalization of results for potential variation in DNA recovery. Availability of this assay should facilitate studies of basic pathogenesis and clinical evaluation of HTLV-I and HTLV-II infection, as well as assessment of therapeutic approaches.

Cyanovirin-N binds to gp120 to interfere with CD4-dependent human immunodeficiency virus type 1 virion binding, fusion, and infectivity but does not affect the CD4 binding site on gp120 or soluble CD4-induced conformational changes in gp120.

Cyanovirin-N (CV-N), an 11-kDa protein isolated from the cyanobacterium Nostoc ellipsosporum, potently inactivates diverse strains of human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus. While it has been well established that the viral surface envelope glycoprotein gp120 is a molecular target of CV-N, the detailed mechanism of action is of further interest. We compared matched native and CV-N-treated virus preparations in a panel of assays that measure viral replication, assessing successive stages of the viral life cycle. CV-N-treated virions failed to infect cells as detected by p24 production and quantitative PCR for HIV-1 reverse transcription products, whereas treatment of the target cells did not block infection, confirming that CV-N acts at the level of the virus, not the target cell, to abort the initial infection process. Compared to native HIV-1 preparations, CV-N-treated HIV-1 virions showed impaired CD4-dependent binding to CD4(+) T cells and did not mediate "fusion from without" of CD4(+) target cells. CV-N also blocked HIV envelope glycoprotein Env-induced, CD4-dependent cell-cell fusion. Mapping studies with monoclonal antibodies (MAbs) to defined epitopes on the HIV-1 envelope glycoprotein indicated that CV-N binds to gp120 in a manner that does not occlude or alter the CD4 binding site or V3 loop or other domains on gp120 recognized by defined MAbs and does not interfere with soluble CD4-induced conformational changes in gp120. Binding of CV-N to soluble gp120 or virions inhibited subsequent binding of the unique neutralizing MAb 2G12, which recognizes a glycosylation-dependent epitope. However, prior binding of 2G12 MAb to gp120 did not block subsequent binding by CV-N. These results help clarify the mechanism of action of CV-N and suggest that the compound may act in part by preventing essential interactions between the envelope glycoprotein and target cell receptors. This proposed mechanism is consistent with the extensive activity profile of CV-N against numerous isolates of HIV-1 and other lentiviruses and supports the potential broad utility of this protein as a microbicide to prevent the sexual transmission of HIV.

Strict conservation of the retroviral nucleocapsid protein zinc finger is strongly influenced by its role in viral infection processes: characterization of HIV-1 particles containing mutant nucleocapsid zinc-coordinating sequences.

The retroviral nucleocapsid (NC) protein contains highly conserved amino acid sequences (-Cys-X2-Cys-X4-His-X4-Cys-) designated retroviral (CCHC) Zn2+ fingers. The NC protein of murine leukemia viruses contains one NC Zn2+ finger and mutants that were competent in metal binding (CCCC and CCHH) packaged wild-type levels of full-length viral RNA but were not infectious. These studies were extended to human immunodeficiency virus type 1 (HIV-1), a virus with two NC Zn2+ fingers. Viruses with combinations of CCHC, CCCC, and CCHH Zn2+ fingers in each position of HIV-1 NC were characterized. Mutant particles contained the normal complement of processed viral proteins. Four mutants packaged roughly wild-type levels of genomic RNA, whereas the remaining mutants packaged reduced levels. Virions with mutated C-terminal position NC fingers were replication competent. One interesting mutant, containing a CCCC Zn2+ finger in the N-terminal position of NC, packaged wild-type levels of viral RNA and showed approximately 5% wild-type levels of infectivity when examined in CD4-expressing HeLa cells containing an HIV-1 LTR/beta-galactosidase construct. However, this particular mutant was replication defective in H9 cells; all other mutants were replication defective over the 8-week course of the assay. Two long terminal repeat viral DNA species could be detected in the CCCC mutant but not in any of the other replication-defective mutants. These studies show that the N-terminal Zn2+ finger position is more sensitive to alterations than the C-terminal position with respect to replication. Additionally, the retroviral (CCHC) NC Zn2+ finger is required for early infection processes. The evolutionary pressure to maintain CCHC NC Zn2+ fingers depends mainly on its function in infection processes, in addition to its function in genome packaging.