Influence of MHC class I and II haplotypes on the experimental infection of Mauritian cynomolgus macaques with SHIVSF162P4cy.

Mauritian cynomolgus macaques (MCM) are widely used in human immunodeficiency virus research because of their restricted major histocompatibility complex (MHC) diversity which provides the opportunity to address the influence of host factors on vaccine studies. We herein report the impact of MHC haplotype on the outcome of 21 MCM infections with the CCR5-tropic simian/human immunodeficiency virus (SHIV)(SF162P4cy). MCM were susceptible to SHIV(SF162P4cy) infection as shown by viremia and loss of CD4+ T cells. A significant association between haplotype M7 (class IA, IB, II) and persistent viremia was observed in chronic phase, whereas recombinant class IA haplotype was associated with a reduction of viral RNA during acute infection. Class IB M4 haplotype displayed significantly lower acute phase provirus copy numbers. In addition, statistical analysis indicated a detrimental effect of haplotype M4 (class IA, IB) on the course of infection as indicated by lower CD4+ T-cell levels during chronic infection. A decrease in post-acute phase CD4+ T-cell numbers was also observed in haplotype M2 animals. This is the first report that documents the effects of host MHC class I and II molecules on the SHIV(SF162P4cy) infection in MCM, particularly with regard to the association between recombinant class IA, M4, and M7 haplotypes and the dynamic of viral replication and level of CD4+ T cells.

Control of SHIV-89.6P-infection of cynomolgus monkeys by HIV-1 Tat protein vaccine.

Vaccine strategies aimed at blocking virus entry have so far failed to induce protection against heterologous viruses. Thus, the control of viral infection and the block of disease onset may represent a more achievable goal of human immunodeficiency virus (HIV) vaccine strategies. Here we show that vaccination of cynomolgus monkeys with a biologically active HIV-1 Tat protein is safe, elicits a broad (humoral and cellular) specific immune response and reduces infection with the highly pathogenic simian-human immunodeficiency virus (SHIV)-89.6P to undetectable levels, preventing the CD4+ T-cell decrease. These results may provide new opportunities for the development of a vaccine against AIDS.

Effects of in vivo Friend leukemia virus infection on levels of serum thymic factors and on selected T-cell functions in mice.

The levels of serum thymic factor(s) (STF), of Thy-1.2 positivity of splenocytes [as measured by their azathioprine (AZ) sensitivity], and of Thy-1.2-positive "spontaneous" spleen rosette-forming cells (SSRFCs), as well as the presence of infectious virus in the thymus, were assessed as a function of time after virus inoculation in susceptible DBA/2, partially resistant BALB/c, and fully resistant C57BL/6 mice given the polycythemia- or anemia-inducing strain of Friend leukemia virus (FLV-P and FLV-A, respectively). As early as Days 2 to 3, the levels of STF and of AZ sensitivity of splenocytes were profoundly decreased in DBA/2 mice, and, to a lesser extent, in BALB/c mice given FLV-P; however, SSRFCs/spleen were increased in both mouse strains. Conclusive evidence of infectious FLV-P was obtained in the thymuses of DBA/2 mice soon after infection. In mice of the same strains infected with FLV-A, STF levels were similarly decreased, but AZ sensitivity of splenocytes was unaffected, and SSRFCs were decreased. Evidence of early FLV-A infection in the thymus of DBA/2 mice was likewise obtained. In C57BL/6 mice given FLV-A, STF levels, AZ sensitivity of splenocytes, and SSRFC showed changes similar to, but of lower magnitude than, those in BALB/c mice. On the other hand, in C57BL/6 mice given FLV-P, the decrease in STF and AZ sensitivity was almost as pronounced as in susceptible DBA/2 mice in the face of complete absence of infectious virus or viral markers in the thymuses. The observed changes are ascribed to virus infection in view of the following: (a) good temporal correlation between these changes and virus infection; (b) absence of any change in mice given heat-inactivated viruses or spleen homogenate of normal DBA/2 mouse spleen; (c) overall good correlation between mouse genotype and genetic (Fv-1 and Fv-2) restrictions of virus infection on one hand and the magnitude of the observed changes on the other. In particular, the decrease in STF and SSRFC levels is ascribed to the replication-competent (Friend-murine leukemia virus) component of Friend leukemia virus complex, whereas the decrease in AZ sensitivity of splenocytes and the increase of SSRFCs are ascribed to the defective spleen focus-forming virus component of the complex. All changes described so far were transient, since they were not detectable beyond 42 days after virus inoculation in overtly leukemic animals. The observed derangements of thymus-derived immune functions may play an important cofactor role during the onset of leukemia in mice genetically permissive to Friend leukemia virus replication and transformation, but they do not seem relevant to the maintenance of leukemia.

HIV, HTLV-1, and HBV infections in a cohort of Italian intravenous drug abusers: analysis of risk factors.

A seroepidemiological survey of a group of 291 intravenous drug abusers (IVDAs), 45 household contacts of IVDAs, and 39 laboratory workers has been carried out to determine the prevalence of HIV-1, HIV-2, HTLV-1, and HBV antibodies in the sera, as well as to evaluate the role of various risk factors. Among i.v. drug abusers, the prevalence was 32.3% for HIV-1 and 6.6% for HTLV-1. For both viruses, the total figures did not significantly change from 1985 through 1987, accounting for a slow viral circulation in this group. No seropositivity (HIV-1, HTLV-1) was found among laboratory workers, whereas one subject was found seropositive for HIV-1 among household contacts. From 1985 to 1986, 5 out of 58 subjects seronegative for HIV-1 and 5 out of 82 seronegative for HTLV-1 seroconverted (incidence rates of 8.6 and 6.1%, respectively). From 1986 to 1987, none out of 11 seronegatives for HIV and 1 out of 16 seronegatives for HTLV-1 seroconverted. The total figures for hepatitis B markers were 79.2% among IVDAs, 24.4% among household contacts, and 25.6% among laboratory workers. A significant correlation was found between presence of HBV markers and seropositivity for HIV and HTLV-1. A significant association with HIV-1 seropositivity was found for history of sexual intercourse with HIV-1 seropositive partners and for sexual promiscuity. These data emphasize the important role played by sexual behavior in addition to needle-sharing in the spreading of multiple infections among drug abusers.

In vitro spontaneous production of anti-SIV antibodies is a reliable tool in the follow-up of protection of SIV-vaccinated monkeys.

To assess the reliability of the spontaneous in vitro synthesis of simian immunodeficiency virus (SIV)-specific antibodies as a marker in the monitoring of protection in SIV-vaccinated animals, Macaca fascicularis monkeys were immunized with formalin-inactivated SIVmac251 or SIVmac251/32H, and challenged with human-derived (SIVmac251/32H) or monkey-derived live SIV. As judged by virus isolation and polymerase chain reaction (PCR) techniques, immunized animals were protected against human-derived SIV challenge, and no spontaneous in vitro synthesis of anti-SIV antibody was observed in nonstimulated peripheral blood mononuclear cell cultures over a 4-month follow-up. On the contrary, human cell-grown SIVmac251 immunization did not afford protection against monkey-derived SIV, and all the animals became infected and showed spontaneous in vitro synthesis of anti-SIV antibodies. These data demonstrate that lack of protection in SIV-vaccinated monkeys is strictly associated with PBMC ability of spontaneously produce anti-SIV antibodies in vitro following challenge, and suggest that this parameter might also constitute a reliable marker for monitoring protection in large-scale HIV vaccination and immunotherapy programs.

DNA immunization of mice against SIVmac239 Gag and Env using Rev-independent expression plasmids.

Simian immunodeficiency virus (SIV) structural gene expression, including gag and env, strictly depends on the interaction of the viral posttranscriptional regulator Rev with its target RNA, the Rev-responsive element (RRE). A small RNA element, termed the constitutive transport element (CTE), located in the 3' portion of simian retrovirus 1 (SRV-1) mRNA, can efficiently substitute for the human immunodeficiency virus (HIV) Rev-RRE interaction, and thus render HIV expression and replication Rev independent. We tested the ability of the SRV-1 CTE to drive the expression of SIVmac239 env and gag from subgenomic constructs designed for possible use in vaccine trials. In vitro expression studies showed that when the SRV-1 sequence is coupled to the SIV gag and env mRNAs, it functions in an orientation-dependent fashion, and leads to strong expression of SIV Gag and Env in human and monkey cell lines; levels of CTE-mediated protein expression were similar to those obtained with a functional Rev-RRE system. On the other hand, in murine fibroblast-like cells, SIV Gag and Env were expressed from constructs at relatively high levels even in the absence of Rev-RRE; nevertheless, their expression was increased by the presence of the SRV-1 CTE. As reported previously for HIV, the murine cell lines appeared to be defective for Rev-RRE activity, and required overexpression of Rev to induce a Rev response. Intramuscular injection of the gag-CTE and env-CTE constructs in BALB/c mice resulted in the expression of the corresponding mRNAs, and the production of anti-Gag and anti-Env antibodies, thus suggesting that these vectors might be used for genetic immunization approaches.

Biologic and molecular characterization of producer and nonproducer clones from HUT-78 cells infected with a patient HIV isolate.

HUT-78 cells were infected with a reverse transcriptase (RT)-positive supernatant of a culture of peripheral blood lymphocytes (PBL) from an AIDS patient and then cloned. Of these clones, two have been isolated and characterized. Clone D10 is persistently and productively infected with an HIV variant. The clone F12, in spite of the presence of an integrated full-length HIV provirus, does not release virus particles in the medium. D10 and F12 clones substantially differ in terms of protein pattern; that is, D10 is super-imposable to infected HUT-78 cells, whereas F12 exhibits a decreased uncleaved p55 gag precursor and the presence of uncleaved gp160 and of a unique p19, although they do not show qualitative or quantitative differences in viral RNA synthesis. Restriction patterns of F12 proviral DNA do not show major genomic deletions. These results indicate that F12 clone cells carry an HIV genome with minor mutations that probably affect the correct production of viral proteins at a posttranscriptional level. In addition, the F12 clone is resistant to high-multiplicity superinfection with HIV-1 or HIV-2.

Interference with complement regulatory molecules as a possible therapeutic strategy in HIV infection.

Drugs which inhibit different stages of the HIV infection process, such as cell entry through CD4 and chemokine receptors, production of double stranded DNA from the HIV genome and maturation of newly produced viruses, are now proposed for AIDS therapy. None of these treatments, however, solve the problem of complete HIV eradication and the frequent appearance of mutants displaying drug resistance. We have recently detailed a strategy describing how HIV protects itself from the human complement and propose that interference of this resistance could be a possible target for therapy.

Time-resolved immunofluorescence: a sensitive and specific assay for anti-HIV antibody detection in human sera.

We describe a new immunoassay, time-resolved fluoroimmunoassay (TR-FIA), for detection of anti-HIV antibodies in human sera. This method is based on the use of a crude virus preparation coated on a polystyrene microtitre plate and of a swine anti-human IgG labelled with a rare earth metal, europium, as fluorescent label chelated with EDTA derivatives. A light pulse from a xenon lamp (340 nm) was used to excite the label and after a 400 microseconds delay time the emission fluorescence was counted for 400 microseconds at 613 nm. This cycle was repeated 1000 times during the total counting time of 1 s. TR-FIA presents considerable advantages over other techniques: (a) it avoids time-consuming, expensive and hazardous virus purification steps; (b) it excludes the use of radiotracers or substrates with potential health risks to reveal the reaction; (c) it has high sensitivity and specificity. A total of 475 serum specimens were tested by ELISA and by TR-FIA. The proportions of positivity were 29.6% by ELISA versus 26.7% by TR-FIA. The sensitivity of both systems was 100%. The specificity was 87.5% for ELISA, whereas it reached a value of 99.4% for immunofluorimetric assay.

Anti-nef antibodies and other predictors of disease progression in HIV-1 seropositive injecting drug users.

A cross-sectional and retrospective longitudinal study has been conducted in three Italian infectious disease centres to evaluate the role of anti-nef antibodies and other markers (HIV-1 p24 antigen, p24 Ag; Beta 2-microglobulin, B2-M; and number of CD4+ lymphocytes) as predictors of disease progression in HIV seropositive injecting drug users (IDUs). The selected patients were: 1) HIV-seropositive IDUs in different stages of HIV infection; 2) HIV-seropositive IDUs who had developed AIDS, from whom serial serum samples were available during the asymptomatic stage, and 3) HIV seropositive IDUs who remained asymptomatic through a follow-up period of the same duration as the patients who developed AIDS. Absence of anti-nef antibodies was associated with symptomatic HIV infection. A significant association between the absence of anti-nef antibodies, the presence of p24 Ag, high levels of B2-M, a number of CD4+ lymphocytes less than 500/ml at first visit and disease progression was found. Subjects who were persistently positive for antibody to nef were less likely to develop AIDS than those who were transiently or persistently negative. This difference was statistically significant (p = 0.03). The results of this study show that absence or disappearance of anti-nef antibodies may be used as predictor of disease evolution in HIV seropositive IDUs. This study also confirms the usefulness of other markers, such as p24 Ag, B2-M and number of CD4+ lymphocytes previously shown to be predictive of rapid disease progression for predicting the course of HIV seropositive IDUs.

Longitudinal characterization of CD4, CD8 T-cell subsets and of haematological parameters in healthy newborns of cynomolgus monkeys.

A longitudinal characterization of immune cell subpopulations (lymphocytes, CD4+ and CD8+ cells), of routine haematological parameters and of immunoglobulin serum levels was carried out in newborn Macaca fascicularis starting from 1 week up to 1 year of life. In neonates, the percentage of CD4+ lymphocytes is almost double, while the percentage of CD8+ cells is lower than that found in adult monkeys (> 5-years old). An inverted trend in the percentage of the two T-lymphocyte subpopulations was observed during the weeks following birth, with a progressive increase of circulating CD8+, paralleled by a decrease of CD4+ cell number. Consequently, the CD4/CD8 ratio slowly decreases, even if, at 12 months of life, it is still higher than that found in adult animals. Several differences were also noted between young and adult monkeys with regard to the total number of circulating CD4+ and CD8+ cells. Haematological parameters did not show consistent differences with respect to adult values. The plasma IgG level is high at birth, then decreases until 6 months of life, while the IgM and IgA values are very low during the first weeks of life but increase in the following period. Our data showed that variations of immunological (CD4+, CD8+ cells) patterns and of some haematological parameters in M. fascicularis are dependent on age. These variations should be therefore considered whenever young animals are used in experimental protocols.

Induction of antibodies and T cell responses by a recombinant influenza virus carrying an HIV-1 TatΔ51-59 protein in mice.

Recombinant influenza viruses hold promise as vectors for vaccines to prevent transmission of mucosal pathogens. In this study, we generated a recombinant WSN/TatΔ(51-59) virus in which Tat protein lacking residues 51 to 59 of the basic domain was inserted into the N-terminus of the hemagglutinin (HA) of A/WSN/33 virus. The TatΔ(51-59) insertion into the viral HA caused a 2-log reduction in viral titers in cell culture, compared with the parental A/WSN/33 virus, and severely affected virus replication in vivo. Nevertheless, Tat-specific antibodies and T cell responses were elicited upon a single intranasal immunization of BALB/c mice with WSN/TatΔ(51-59) virus. Moreover, Tat-specific immune responses were also detected following vaccine administration via the vaginal route. These data provide further evidence that moderately large HIV antigens can be delivered by chimeric HA constructs and elicit specific immune responses, thus increasing the options for the potential use of recombinant influenza viruses, and their derivatives, for prophylactic and therapeutic vaccines.

Viral outcome of simian-human immunodeficiency virus SHIV-89.6P adapted to cynomolgus monkeys.

Simian-human immunodeficiency virus (SHIV) 89.6P is considered to be one of the most pathogenic chimeric viruses in rhesus macaques. However, when crossing from one to another species of monkeys the pathogenicity of this virus may be affected. By using SHIV-89.6P(cy243), a virus obtained by passaging SHIV-89.6P in cynomolgus macaques, we investigated the dynamics of viral replication and the impact of the inoculum size (from 10 up to 50 monkey infectious dose) on the progression of the infection in 22 cynomolgus macaques. SHIV-89.6P(cy243 )caused massive depletion of CD4+ T-cells within 4 weeks of the inoculum, followed by an irreversible immune deficiency in a high proportion of the infected monkeys. This study demonstrates that SHIV-89.6P(cy243) is pathogenic in cynomolgus macaques and that the dynamics of the viral replication and the rate of clinical progression depend on the size of the inoculum. Our findings provide unique and relevant data, particularly with regard to the value of the in vivo titration used to select the most appropriate infectious dose to study the "virus-host" interplay.

Homologous superinfection of both producer and nonproducer HIV-infected cells is blocked at a late retrotranscription step.

An HUT-78 cell clone (F12) chronically infected by a nonproducer HIV-1 variant (Federico et al., (1989) AIDS Res. Hum. Retroviruses 5, 385-396) is fully resistant to superinfection with HIV-1 or HIV-2. We demonstrate that, in spite of the down-regulation of CD4 receptors, superinfecting-HIV-1 and -HIV-2 cross the F12 plasma membrane (even in the presence of OKT4A monoclonal antibodies) but fail to complete retrotranscription. We utilized a series of polymerase chain reaction primers designed to detect certain steps in the reverse transcription process. Superinfecting-HIV-1 (an African strain) and -HIV-2 are detectable using primers specific for env (for HIV-1), 5' LTR (the R and U5 regions), and vpx (for HIV-2). No amplification is visible when primers amplifying either HIV-2 gag or the "primer binding site" region of 5' LTR of HIV-2 are used. DNA-PCR performed on DNAse-pretreated HIV-1 and HIV-2 stocks failed to show any amplification. This rules out that any extra- or intravirion viral DNA contamination may have interfered with our results. In addition, no DNA amplification was observed in F12 and HUT-78 cells exposed to heat-inactivated HIV-2. Finally, when the nonproducer F12 cells as well as control CEMss cells are transfected with the HIV-1 infectious molecular clone pNL4-3, progeny infectious virus is obtained. These findings indicate that reverse transcription of HIVs superinfecting F12 cells is prevented from completing viral DNA synthesis. A similar block occurs in HIV-1-infected producer cells. When integration of the HIV genome into the F12 genome is achieved via transfection of a molecular clone, the virus life cycle can proceed as in control CEMss cells.