Jim Stott

'Chemical condoms' for the prevention of HIV infection: evaluation of novel agents against SHIV(89.6PD) in vitro and in vivo.

Background Vaginal agents which are antiviral and/or inhibit the entry of HIV into the cell could prevent heterosexual transmission of HIV, and protect women who cannot negotiate condom use. Methods Four agents have been investigated for activity in vitro and in vivo against SHIV89.6PD: two anionic polymers, dextrin-2-sulphate (D2S) and PRO 2000 (P2K), and two virucidal agents; a non-ionic detergent, nonoxynol-9 (N9) and a cyclic peptide ionophore, gramicidin-D (GD). All four agents were investigated in rhesus macaques, using an intra-vaginal challenge of two inoculations of 1 × 104 50% tissue culture infectious doses (TCID)50of SHIV89.6PD. Results D2S, P2K, GD and N9 all inhibited SHIV89.6PDin vitro. In vivo, three out of four control macaques were infected as judged by viral culture, seroconversion, DNA and RNA PCR; infection was confirmed in four out of eight macaques pre-treated with P2K, two out of four pre-treated with D2S, one out of four pre-treated with N9, two out of four pre-treated with GD and four out of four pre-treated with D2S + GD, a combination additive in vitro. Interpretation D2S and PRO-2000, novel inhibitors of HIV entry, showed evidence of protection in vivo, comparable to that seen with the virucide, N9. These data, together with the results of phase I and phase II studies in healthy women which have shown minimal toxicity, support plans for a phase III efficacy trial of chemically simple inhibitors of HIV entry with low toxicity, for the prevention of HIV infection in women.

CD8+ Lymphocytes Do Not Mediate Protection against Acute Superinfection 20 Days after Vaccination with a Live Attenuated Simian Immunodeficiency Virus

ABSTRACT In order to test the hypothesis that CD8+ cytotoxic T lymphocytes mediate protection against acute superinfection, we depleted >99% of CD8+ lymphocytes in live attenuated simian immunodeficiency virus macC8 (SIVmacC8) vaccinees from the onset of vaccination, maintained that depletion for 20 days, and then challenged with pathogenic, wild-type SIVmacJ5. Vaccinees received 5 mg per kg of humanized anti-CD8 monoclonal antibody (MAb) 1 h before inoculation, followed by the same dose again on days 3, 7, 10, 13, and 17. On day 13, peripheral CD8+ T lymphocytes were >99% depleted in three out of four anti-CD8 MAb-treated vaccinees. At this time attenuated SIVmacC8 viral RNA loads in anti-CD8 MAb-treated vaccinees were significantly higher than control vaccinees treated contemporaneously with nonspecific human immunoglobulin. Lymphoid tissue CD8+ T lymphocyte depletion was >99% in three out of four anti-CD8 MAb-treated vaccinees on the day of wild-type SIVmacJ5 challenge. All four control vaccinees and three out of four anti-CD8 MAb-treated vaccinees were protected against detectable superinfection with wild-type SIVmacJ5. Although superinfection with wild-type SIVmacJ5 was detected at postmortem in a single anti-CD8 MAb-treated vaccinee, this did not correlate with the degree of preceding CD8+ T lymphocyte depletion. Clearance of attenuated SIVmacC8 viremia coincided with recovery of normal CD8+ T lymphocyte counts between days 48 and 76. These results support the view that cytotoxic T lymphocytes are important for host-mediated control of SIV primary viremia but do not indicate a central role in protection against acute superinfection conferred by inoculation with live attenuated SIV.

Resistance to superinfection by a vigorously replicating, uncloned stock of simian immunodeficiency virus (SIVmac251) stimulates replication of a live attenuated virus vaccine (SIVmacC8)

Vaccination with live attenuated simian immunodeficiency virus (SIVmacC8) confers potent, reproducible protection against homologous wild-type virus challenge (SIVmacJ5). The ability of SIVmacC8 to confer resistance to superinfection with an uncloned ex vivo derivative of SIVmac251 (SIVmac32H/L28) was investigated. In naïve, Mauritian-derived cynomolgus macaques (Macaca fascicularis), SIVmac32H/L28 replicated to high peak titres (>10(8) SIV RNA copies ml(-1)), persisted at high levels and induced distinctive pathology in lymphoid tissues. In cynomolgus macaques vaccinated with SIVmacC8, no evidence of detectable superinfection was observed in 3/8 vaccinates following challenge 3 or 20 weeks later with SIVmac32H/L28. Analyses after SIVmac32H/L28 challenge revealed a significant reduction in viral RNA (P<0.001) and DNA levels between 20 week vaccinates and challenge controls. Amongst 3 week vaccinates, less potent protection was observed. However, analysis of env from breakthrough virus indicated >99% sequence similarity with the vaccine virus. Highly sensitive PCR assays that distinguish vaccine and challenge virus stocks demonstrated restimulation of replication of the vaccine virus SIVmacC8 in the face of potent protection against a vigorous, homologous challenge virus. Vaccine-induced antiviral neutralizing antibodies and anti-Nef CD8+ cytotoxic T cell responses did not correlate with the outcome of the challenge. Defining the mechanism of vaccine protection will need to account for the effective control of a genetically closely related challenge virus whilst remaining unable to suppress replication of the pre-existing vaccine virus. The role of innate and intrinsic anti-retroviral immunity in the protection conferred by live attenuated SIV vaccines warrants careful study.

Protection of macaques against simian immunodeficiency virus infection with inactivated vaccines: comparison of adjuvants, doses and challenge viruses

Nine European laboratories contributed a total of 98 macaques towards a collaborative trial to study the ability of formaldehyde-inactivated or subunit SIV vaccines to protect immunized animals against live virus challenges. Four adjuvants, three dose levels and two immunization schedules were compared. Fifty-two of 61 (85%) immunized animals were protected against infection after challenge with either homologous or heterologous virus strains grown in human cells. Optimum protection required a high dose of antigen and a prolonged immunization schedule. On the day of challenge the titres of antibodies to SIV and to host cell components, as well as the titres of neutralizing antibodies, were significantly higher in the protected animals than in the non-protected. Forty-four vaccinated macaques (of which 36 were protected against previous challenges grown in human cells) and 28 naive animals were then challenged with extracellular or cell-associated SIV grown in simian cells. All naive animals and all vaccinees challenged with extracellular SIV became infected. Four of the eight animals challenged with cell-associated viruses were protected. These results clearly indicate that vaccines which potently protect against SIV grown in human cells, do not protect against SIV grown in simian cells. The cell substrate on which challenge viruses are grown is clearly significant in interpreting the results of vaccine trials. This trial has demonstrated that SIV vaccines using different adjuvants can protect macaques against SIV grown in human cells but not against extracellular SIV grown in simian cells. These results have important relevance to the development of HIV vaccines for humans.(ABSTRACT TRUNCATED AT 250 WORDS)

LIVE ATTENUATED SIV : A MODEL OF A VACCINE FOR AIDS

The experimental infection of macaques with simian immunodeficiency virus (SIV) has provided strong evidence that it may be possible to develop a vaccine against AIDS. Live attenuated SIV vaccines have been found to confer the most potent protection against challenge with a variety of pathogenic viruses. This article summarizes the work performed at NIBSC to characterize the protection conferred by live attenuated SIV and to identify mechanisms of vaccine protection. The results of these experiments are discussed in conjunction with observations from related studies made by other groups.

Mechanisms of protection induced by live attenuated simian immunodeficiency virus: III. Viral interference and the role of CD8+ T-cells and beta-chemokines in the inhibition of virus infection of PBMCs in vitro.

In this study, we investigated whether a type of retroviral interference might be one mechanism that mediates the powerful protection induced by live attenuated SIVC8. Our results show that retroviral interference could be demonstrated between SIV and SHIV‐HXBc2 in human T‐cell lines chronically infected with either SIVC8 or SIVJ5. Lymphocytes from macaques infected with live attenuated SIVC8 were significantly less sensitive (P<0.05) to in vitro infection by virulent SIVJ5 and SHIV‐HXBc2 than were lymphocytes from naïve controls. However, this significant difference in the sensitivity of lymphocytes to virus infection was not observed for more efficiently replicating viruses such as SHIVSF33 and SIVsm3. Virus growth was significantly enhanced (P<0.01) by depletion of CD8+ T‐cells, suggesting a role for these cells in the control of SIV replication, both in vitro and in vivo. We found that levels of the β‐chemokines regulated upon activation, normal T‐cell expressed and secreted, macrophage inflammatory protein‐1α and macrophage inflammatory protein‐1β did not correlate with inhibition of virus replication. Taken together, our findings do not support the hypothesis that retroviral interference is the mechanism by which live attenuated SIVC8 induces protection.

Specific Proliferative T Cell Responses and Antibodies Elicited by Vaccination with Simian Immunodeficiency Virus Nef Do Not Confer Protection against Virus Challenge

The efficacy of immunizing with a combination of simian immunodeficiency virus (SIV) Nef vaccines was evaluated. Four vaccinates received three intradermal immunizations with recombinant vaccinia virus that expressed SIV Nef, followed by three intramuscular immunizations with rDNA also expressing SIV Nef. Finally, the four vaccinates received two subcutaneous boosts with recombinant SIV Nef protein. This immunization protocol elicited anti-Nef antibodies in all of the vaccinates as well as specific proliferative responses. However, specific cytotoxic T cell responses were not detected before virus challenge. All vaccinates were challenged intravenously with 10 MID(50) of SIVmacJ5 along with four controls. All eight subjects became infected after SIV challenge and there were no group-specific differences in virus load as measured by virus titration and vRNA analysis. The results of this study support indirectly the report from Gallimore and colleagues (Nat Med 1995;1:1667) suggesting that CD8(+) T lymphocyte responses are required for Nef-based vaccines to restrict SIV infection. If Nef-based vaccines are to be beneficial in controlling infection with immunodeficiency viruses, then it will be necessary to develop more effective immunization protocols that elicit potent CD8(+) cell responses reproducibly.

Immunological responses and viral modulatory effects of vaccination with recombinant modified vaccinia virus Ankara (rMVA) expressing structural and regulatory transgenes of simian immunodeficiency virus (SIVmac32H/J5M)

Background  The immunogenicity and protective efficacy of recombinant modified vaccinia virus Ankara (rMVA) vectors expressing structural (gag/pol, env) and regulatory (tat, rev, nef) genes of SIVmac251/32H‐J5 (rMVA‐J5) were assessed.