GENERATION OF AN AIDS-INDUCING R5-TROPIC SHIV WITH NOVEL PATHOGENIC PROPERTIES BY SERIAL PASSAGING IN RHESUS MACAQUES We have generated a new pathogenic R5-tropic SHIV following long-term animal-to-animal passage, which bears the env gene from the prototypical macrophage tropic strain, HIV 1Ada. SHIVAD8 is able to sustain plasma viremia in rhesus monkeys for more than 2 years;all 13 inoculated animals experienced marked depletions of CD4+ T lymphocytes. Ten of these monkeys became normal progressors (NPs), generated anti-viral CD8+ T cell responses, and sustained chronic immune activation. Several of these animals also produced high titers of NAbs during the course of their infections. The most consistent and distinguishing in vivo property of the SHIVAD8 family of viruses is the slow and unremitting loss of both memory and nave CD4+ T cells. This pattern of depletion was observed in all 10 NPs. During the chronic phase of infection, the loss of nave CD4+ T cells was more rapid and marked than the depletion of the memory subset. By week 80, NPs had sustained an 87 to 93% loss of nave cells from their pre-inoculation levels. An assessment of coreceptor utilization of late stage viruses recovered from SHIVAD8 NPs indicated that a coreceptor switch had not occurred and the virus present throughout the infection had remained R5 tropic. Surprisingly, and in contrast to both SIVmac and SIVsmE lineages, the pace of CD4+ T lymphocyte decline did not correlate with plasma virus loads. Although the geometric mean plasma viral RNA level, at week 50 in the SHIVAD8 infected monkey cohort was 1.7 x 10e3 RNA copies/ml, the set-point virus loads varied widely in different animals (1.6 x 10e2 to 1.5 x 10e5 RNA copies/ml). Thus far, 7 of the 10 SHIVAD8 NPs have developed clinical symptoms of immunodeficiency requiring euthanasia between weeks 100 and 117 PI. These monkeys sustained opportunistic infections involving Mycobacterium avium, Pneumocystis carinii, and Campylobacter coli. In addition, two other NPs have lost a significant amount of weight, are experiencing chronic diarrhea, and currently have total circulating CD4+ T cell counts of 39 and 75 cells/μl, respectively. WHAT LEVEL OF CIRCULATING NEUTRALIZING ANTIBODIES CONFER PROTECTION TO MACQUES FROM A SUBSEQUENT SHIV CHALLENGE? The development of an effective vaccine against the human immunodeficiency virus (HIV) is critical for controlling the acquired immunodeficiency syndrome (AIDS) epidemic. A variety of assays have been employed to measure the anti-viral cellular and humoral responses elicited by these vaccines to delineate correlates of protection. The results from studies involving both SIVs and SHIVs have shown that pre-challenge cellular responses alone do not prevent virus acquisition, although they can reduce the peak and set point levels of viremia. The contribution of vaccine-induced neutralizing antibodies (NAbs) in controlling virus infection has been more difficult to evaluate. Measurements of the level and breadth of NAb responses in samples collected from vaccinees, infected individuals, or HIV surrogate animal models have relied on a variety of anti-viral neutralization assays. Early assays relied on the control of spreading virus infections, requiring that cultures be maintained for extended periods of time for the anti-viral neutralization effects to become evident. The development and use of the TZM-bl cell based assay has greatly simplified measurements and reduced inter-laboratory variability in monitoring anti-HIV 1 NAbs. Despite the plethora of data generated with the TZM-bl system, the assay has not yet been used to determine virus neutralization titers that confer protection to individuals or animals exposed to virus. We previously administered purified IgG containing high titered polyclonal NAbs against SHIVDH12 to 21 pigtailed macaques and determined the NAb titer (1:38) in the plasma required to protect 99% of animals against an intravenous (IV) challenge of 75 TCID50 of virus ( ). This protective titer was derived from a 14-day MT4 cell assay, which measures the complete inhibition (EC100) of SHIVDH12 replication in cell cultures at the limiting dilution end-point. The TZM-bl system was used to measure EC50 neutralizing titers in several of the same macaque plasma samples and augmented probit regression was employed to determine EC50 titers conferring various levels of protection. When psudotyped virus was used in the TZM-bl assay, it was determined that 99% protection would require an EC50 titer of 1:4467 (not 1:38 as measured in the MT4 assay). Because it is likely that contributions from other arms of the immune system would contribute to vaccine-induced control, a range of protective EC50 NAb titers for this data set were also derived: 99% / 1:4467;90% / 1:1175;80% / 1:676;50% / 1:234;and 33% / 1:141. This range of titers may be more informative for evaluating the protective value of NAb activities determined on patient samples using the TZM bl assay.
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