Titration of a vaccine stock preparation of human immunodeficiency virus type 1SF2 in cultured lymphocytes and in chimpanzees.

A large stock preparation of the HIV-1SF2 isolate has been derived after serial passage in human peripheral blood mononuclear cells (PBMCs). This viral stock has a titer of 10(4.9) TCID50 in human PBMCs and 10(4.2) TCID50 in chimpanzee PBMCs. By inoculation into animals the 50% chimpanzee infectious dose titer was found to be about 10(2.3). Virus isolation from animals was achieved on most occasions within 1-4 weeks after inoculation and then became transient. Viral RNA and DNA PCR analyses confirmed the virus infection of the chimpanzees. Anti-HIV antibody levels in the inoculated animals ranged from 1:400 to 1:6400 as measured by ELISA. About 680 vials of this stock preparation, frozen at -190 degrees C, are available for future studies of vaccines and antiviral therapies.

Resistance of chimpanzees immunized with recombinant gp120SF2 to challenge by HIV-1SF2.

OBJECTIVE: To determine whether vaccination with recombinant HIV-1SF2 gp120 in a novel oil-in-water adjuvant emulsion, MF59, protects chimpanzees against challenge with HIV-1SF2, the homologous virus isolate. METHODS: Two vaccinated chimpanzees and two control animals were challenged with 25-50 animal infectious doses of a stock of HIV-1SF2 that had been grown in mitogen-activated human peripheral blood mononuclear cells (PBMC). The animals were monitored by a series of serologic [enzyme-linked immunosorbent assay (ELISA), Western blot, and neutralization assays] and virologic [virus culture, RNA and DNA polymerase chain reaction (PCR)] assays for infection. RESULTS: Both control animals showed evidence of seroconversion in ELISA and Western blot assays. In addition, virus was detected in the early, acute phase of infection of both control animals by (1) plasma RNA PCR, (2) virus culture, and (3) PBMC DNA PCR assays. One vaccinated animal showed no serologic or virologic evidence of infection. The other vaccinated animal has not seroconverted, and there was no evidence of plasma viremia. However, virus was detected at early timepoints in this animal's PBMC, and transient lymphoproliferation to HIV-1 proteins not in the vaccine was observed. These observations suggest that the former animal was protected from challenge while the latter may have experienced a transient or curtailed infection. CONCLUSION: Two types of vaccine-induced protective immune responses were observed when chimpanzees immunized with rgp120SF2 were challenged with the homologous virus isolate: a response consistent with the 'sterilizing immune response' documented in the chimpanzee model in previous studies, as well as one that did not completely protect from infection, showing curtailment of the acute phase and a failure of the animal to seroconvert.

Reduced virus load in rhesus macaques immunized with recombinant gp160 and challenged with simian immunodeficiency virus.

As a safe alternative to inactivated and live-attenuated whole-virus SIV vaccines, we have evaluated the potential of SIVmac239 gp160 expressed by recombinant vaccinia virus (vSIVgp160) and baculovirus (bSIVgp160) to protectively immunize rhesus macaques against intravenous (i.v.) infection with pathogenic SIVmac isolates. Macaques were immunized with live vSIVgp160 and/or bSIVgp160 protein partially purified from insect cells. The challenge viruses, propagated in rhesus peripheral blood mononuclear cells, consisted of the molecular clone SIVmac239 and another genetically similar, uncloned isolate, SIVmac251. Although antibodies that bind gp130 were induced in all animals following immunization with SIVgp160, neutralizing antibodies were undetectable 1 week prior to virus challenge. These results differ from those for macaques vaccinated with inactivated, whole SIV. All animals became infected after i.v. inoculation with 1-10 AID50 of either challenge virus. For animals challenged with SIVmac251, but not those challenged with SIVmac239, the cell-free infectious virus load in plasma of vSIVgp160-primed, bSIVgp160-boosted macaques was significantly lower than in unimmunized controls at 2 weeks postchallenge. Virus virulence, immunization regimen, and challenge with homologous or heterologous virus are factors critical to the outcome of the study. Immunization with surface glycoprotein may not necessarily provide protective immunity against infection but may reduce virus load. The relationship between reduction in virus load by vaccination and delay in onset of disease remains to be determined.

Absence of HIV-1 DNA in high-risk seronegative individuals using high-input polymerase chain reaction.

Evidence of frequent HIV-1 infections in antibody-negative, high-risk individuals (so-called 'silent' infections) remains controversial. To evaluate whether these discrepant results may be the consequence of intermittent detection of rare infected cells (low viral load) preceding seroconversion, we developed a modification of the polymerase chain reaction (PCR) technique which enabled analysis of 10-fold greater amounts of cellular DNA per reaction than standard PCR (2 x 10(6) rather than 0.2 x 10(6) input cells). This technique allowed consistent detection of HIV-1 provirus in two seropositive individuals who had repeatedly tested negative by standard-input PCR. However, results were negative when high-input PCR was applied to 51 specimens from 39 selected high-risk seronegative individuals. These results suggest that variations in viral load preceding or in the absence of seroconversion probably do not explain discrepant evidence regarding silent HIV-1 infection.

Reliable confirmation and quantitation of human immunodeficiency virus type 1 antibody using a recombinant-antigen immunoblot assay.

The recombinant DNA-derived, human immunodeficiency virus (HIV) antigen-based immunoblot assay (RIBA-HIV216) is a new supplemental (confirmatory) test developed to detect antibodies to HIV-1. The assay employs four recombinant viral antigens, corresponding to HIV-1 p24, p31, p41 and gp120 proteins, in an immunoblot format. With this assay, HIV-1 antigen reactivity was detected in all 683 infected patient serum or plasma specimens evaluated; 665 (97.6%) of these sera met the criteria for a positive interpretation, and 18 (2.6%) were classified as indeterminate. All 683 samples reacted with the recombinant gp41-equivalent protein. The first sequential enzyme immunoassay (EIA)-reactive samples collected from 33 seroconverting homosexual men reacted on RIBA-HIV216. Eleven (1.1%) of 999 EIA-negative blood donor sera reacted weakly with a single recombinant antigen (p24 or p31), whereas 13 to 48 percent had indeterminate reactions on viral lysate Western blots. One (1.5%) of 66 EIA-positive, Western blot-negative blood donor samples and 19 (29%) of 66 EIA-positive, Western blot-indeterminate blood donor samples scored indeterminate on RIBA-HIV216. Nonspecific reactivity was seen with only 1 (0.8%) of 114 patient sera containing possible interfering antibodies, whereas 33 percent of these samples had indeterminate reactions on Western blot and 35 percent had equivocal reactions on immunofluorescence assay (IFA). We conclude that the RIBA-HIV216 is approximately as sensitive as and significantly more specific than virus-derived Western blot and IFA. The RIBA-HIV216 also allows for semiquantitation of specific antibodies that may be of value in clinical staging and therapeutic monitoring.