Rapid communication: development of in vitro resistance to macrophage-tropic- and T-cell-line-adapted HIV-1 strains among HIV-positive volunteers treated with highly active antiretroviral therapy.

We have described a peripheral blood mononuclear cell (PBMC) culture system in which control of endogenous virus and resistance to exogenous HIV-1 correlates with low viremia among HIV-1-positive people. Nineteen patients remained consistently resistant or susceptible for more than 5 years of follow-up. On the fifth year, 5 consistently susceptible volunteers with high viral loads began receiving highly active anti-retroviral therapy (HAART). After >6 months on HAART, 5 of 5 became completely or predominantly resistant on four visits over the next 6 months. Among HIV-1-positive donors, we had never observed reversal of PBMC phenotype from consistently susceptible to consistently resistant. Resistance correlated with suppression of plasma viremia and rebound in CD4+ T-cell counts and percentages. When resistant PBMCs were challenged after CD8+ T-cell depletion, 38 of 41 and 40 of 59 cultures became susceptible to HIV-1MN and HIV-1BaL, respectively. After combined CD8+ T-cell depletion and antibody neutralization of beta-chemokines, 16 of 18 cultures became susceptible to HIV-1BaL. Overall, the finding that >90% of these cultures depleted of relevant antiviral effector arms could become infected indicates resistance was not due to residual antiretroviral drug metabolites in vitro. For 2 volunteers who discontinued therapy because of side effects, pretreatment viral load correlated with loss of in vitro resistance and viral rebound. In addition to resistance to laboratory strains of HIV-1, all patients developed resistance to at least one of two CCR5-tropic, clade B primary isolates: HIV-1P15 and HIV-1P27.

Resistance to human immunodeficiency virus type 1 in vitro as a surrogate of vaccine-induced protective immunity.

An in vitro assay developed as a correlate of vaccine-induced protection from human immunodeficiency virus (HIV) was validated in populations with relative resistance to HIV-1 as well as in HIV vaccine recipients. Cultures of peripheral blood mononuclear cells (PBMC) were challenged with 10 TCID50 of HIV-1MN or HIV-1BaL, titered in PBMC from normal controls (n=57). PBMC from HIV-1-infected persons with low viremia (n=17), exposed uninfected persons (n=23), and HIV-2-infected Senegalese prostitutes (n=9) were significantly resistant to HIV-1BaL and/or HIV-1MN (P<.001). Among 34 HIV vaccine recipients of live canarypox vector expressing multiple HIV-1 gene products with or without rgp120 booster, PBMC from postvaccination samples were significantly resistant to both strains (P<.001), and cytotoxic T lymphocyte precursor-positive samples were significantly more resistant than were precursor-negative samples (P<.03). This is the first evidence of the induction by vaccination of a validated correlate of protection. This assay should serve as a useful criterion for assessing experimental HIV vaccines before phase III efficacy trials.

A novel factor produced by placental cells with activity against HIV-1.

The factors controlling the dynamics of HIV-1 transmission from mother to infant are not clearly known. Previous studies have suggested the existence of maternal and placental protective mechanisms that inhibit viral replication in utero. Preliminary studies from our laboratory revealed that supernatant from placental stromal cells protected HIV-1-infected PBMC from virus-induced apoptosis and suppressed virus production. We have attempted to characterize the antiviral activity of this placental factor (PF) and delineate the stages of HIV-1 replication affected. This activity was not due to the presence of any known cytokine reported to have anti-HIV effect. Direct exposure to PF had no suppressive effect on the infectivity of cell-free HIV-1, and envelope-mediated membrane fusion appeared to be unaffected. Western blot analysis of HIV-1 from infected PBMC treated with PF revealed that expression of all viral proteins was reduced proportionately, both intracellularly and in released virions. However, exposure of HIV-1-infected cells to PF resulted in production of virions with 10-100-fold-reduced infectivity. PF-treated virions contained two- to threefold reduced ratios of cyclophilin A:Gag protein as compared with untreated virus. Reduced cyclophilin A content resulting in decreased binding of cyclophilin A to Gag could account, in part, for the observed reduction in infectivity. Our results suggest that placental cells produce an antiviral factor that protects the fetus during gestation and may have therapeutic potential.

Chemokine-independent in vitro resistance to human immunodeficiency virus (HIV-1) correlating with low viremia in long-term and recently infected HIV-1-positive persons.

Chemokines have been implicated as protective factors against human immunodeficiency virus (HIV) infection, competing for binding to receptors that also function as coreceptors for HIV. In this study of HIV-positive donors, peripheral blood mononuclear cell (PBMC) culture resistance to endogenous and exogenous HIV correlated with low plasma viremia and high in vitro RANTES production. However, resistant cells were not rendered susceptible by neutralization of C-C chemokines, and addition of C-C chemokines did not consistently suppress endogenous virus or exogenous HIV-1MN. In contrast, CD8 T cell depletion markedly decreased the frequency of resistant cultures without reducing C-C chemokine production. Among newly infected persons, half exhibited phenotype switching from preinfection susceptibility to postinfection resistance, suggesting that genetically predetermined constitutive cytokine production or allelic receptor expression are not generally responsible for in vitro resistance and nonprogression.

Extensive evaluation of a seronegative participant in an HIV-1 vaccine trial as a result of false-positive PCR.

BACKGROUND: In the USA, more than 2000 volunteers have received one or more experimental HIV-1 vaccines in phase I and II clinical trials, and there have been breakthrough HIV-1 infections among participants receiving vaccine and placebo. Serological diagnosis of new HIV-1 infections in vaccine-trial participants will become increasingly complicated as more viral components are used in vaccines. Recognition of this problem has led to a reliance on viral-genome measurement to distinguish vaccine-induced immunity from HIV-1 infection. Currently, quantitative RNA measurement is expensive, prone to contamination, and reliable only in laboratories certified by manufacturers or that have quality-control programmes. METHODS: A high-risk vaccinee presented after an acute febrile episode and was tested for HIV-1 infection by reverse transcriptase (RT) PCR of viral RNA. Further extensive tests were required to clarify the HIV-1 infection and immune status of the vaccinee, including repeat RT-PCR, nested DNA PCR, western blot, lymphoproliferation assay, cytotoxic T-cell lysis, CD8-depleted co-culture, and HIV-1 challenge culture. FINDINGS: Initial testing of plasma by RNA RT-PCR was reported as positive. This result was not confirmed by viral cultures, nested DNA PCR, western blot, or EIA. Additional RNA RT-PCR assays gave positive results from earlier occasions in the vaccine trial. Eventually, testing of all previously reactive samples by RNA RT-PCR was repeated in a quality-controlled laboratory, and confirmed the negative HIV-1 status of the individual. INTERPRETATION: This case report exemplifies the difficulties with use of viral-genome measurement as a screening test to diagnose HIV-1 infection, particularly in individuals who have ever participated in HIV-1 vaccine trials. Monitoring of large numbers of phase III vaccinees by RNA RT-PCR will not be feasible. The design of efficacy trials for new vaccines should be in parallel with development of antibody-based diagnostic tests that are capable of differentiating between immunisation and true HIV-1 infection.

Newborn rats in the murine typhus enzootic infection cycle: studies on transplacental infection and passively acquired maternal antirickettsial antibodies.

This study focused attention on the newborn rat as a possible significant participant in the highly successful enzootic cycle of murine typhus. We examined the influence of maternal Rickettsia typhi (R. mooseri) infection in rats on the offspring with respect to the possible vertical transmission of R. typhi and the passive transfer of maternal antirickettsial antibodies. Transmission of R. typhi by rickettsemic pregnant rats did not occur either transplacentally during gestation to their fetuses or postnatally through colostrum and milk to their newborn. The rickettsial burden of the placenta was sometimes greater than 10(6) plaque forming units per g tissue and undetectable in colostrum or milk. However, newborn rats were highly susceptible to infection per os. Transplacental passage of antirickettsial antibody to offspring was detectable only when the mother's antibody titer was high. Passive postpartum acquisition of antirickettsial antibodies by newborn rats from colostrum and milk of immune mothers occurred regardless of the height of the maternal antibody titer, rose to a maximum at about 3 weeks of age, and then declined rapidly, becoming undetectable 4 weeks after birth.

Experimental infection with Rickettsia mooseri and antibody response of adult and newborn laboratory rats.

Quantitative studies of selected features of peripherally induced Rickettsia mooseri (= R. typhi) infection in Rattus norvegicus-derived white laboratory rats revealed a unique association between microbe and amplifying vertebrate host which appears to be especially conducive to maintenance of the enzootic cycle. Both adult and newborn (1-3 days old) rats were highly susceptible to percutaneous infection (ID50 = approximately 1 organism), but neither showed signs of disease or died even when inoculated with 10(4)-10(5) plaque-forming units. Gain in body weight of infected newborn rats was indistinguishable from that of uninfected newborn rats over the first 3 weeks of life. The course of the systemic infection, as measured by the rise and fall of R. mooseri titers in blood, brain and kidney and the serum antibody response, was almost identical in adult and newborn rats. Thus, despite their immaturity in certain immunological processes, newborn rats controlled postnatal R. mooseri infection about as well as did adult rats. The rickettsemic period of about 10 days corresponds to the period of infectivity of inoculated rats for fleas. Rickettsiae were not isolated from blood, brain or kidneys by methods employed for more than 4-5 weeks after infection. Serum antirickettsial antibodies persisted for at least 60 weeks postinfection, i.e., longer than the usual life span of rats in nature and, hence, are a valid measure of the cumulative experience of rat populations with R. mooseri infection.