Epidemiological and molecular characterization of HBV and HCV infections in HIV-1-infected inmate population in Italy: a 2017-2019 multicenter cross-sectional study.

HBV/HCV co-infection is common in HIV-1-infected prisoners. To investigate the characteristics of HIV co-infections, and to evaluate the molecular heterogeneity of HIV, HBV and HCV in prisoners, we carried-out a multicenter cross-sectional study, including 65 HIV-1-infected inmates enrolled in 5 Italian detention centers during the period 2017-2019. HIV-1 subtyping showed that 77.1% of inmates were infected with B subtype and 22.9% with non-B subtypes. Italian nationals were all infected with subtype B (93.1%), except two individuals, one infected with the recombinant form CRF72_BF1, and the other with the HIV-1 sub-subtype A6, both previously not identified in inmates of Italian nationality. Non-Italian nationals were infected with subtype B (52.6%), CRFs (36.8%) and sub-subtypes A1 and A3 (5.2%). HIV variants carrying resistance mutations to NRTI, NNRTI, PI and InSTI were found in 7 inmates, 4 of which were never exposed to the relevant classes of drugs associated with these mutations. HBV and/or HCV co-infections markers were found in 49/65 (75.4%) inmates, while 27/65 (41.5%) showed markers of both HBV and HCV coinfection. Further, Italian nationals showed a significant higher presence of HCV markers as compared to non-Italian nationals (p = 0.0001). Finally, HCV phylogenetic analysis performed in 18 inmates revealed the presence of HCV subtypes 1a, 3a, 4d (66.6%, 16.7% and 16.7%, respectively). Our data suggest the need to monitor HIV, HBV and HCV infections in prisons in order to prevent spreading of these viruses both in jails and in the general population, and to implement effective public health programs that limit the circulation of different genetic forms as well as of viral variants with mutations conferring resistance to treatment.

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.

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.

Primary effusion lymphoma cells undergoing human herpesvirus type 8 productive infection produce C-type retroviral particles.

Primary effusion lymphomas (PELs) are invariably infected by the human herpesvirus 8 (HHV8)that is present in most PEL cells as latent virus but replicates in a subset of permissive cells to produce infectious progeny. Here we show that productively infected PEL cells release C-type retrovirus-like particles encoding an Mn++-dependent RT activity, which is typical of endogenous retroviruses. Strikingly, C-type particles are produced only in cells showing advanced HHV8 morphogenesis. Phorbol esters, which induce productive HHV8 replication and morphogenesis in PEL cells, increase RLP production. Phosphonoacetic acid, a blocker of HHV8 late gene expression, inhibits the production of C-type particles, whereas neutralizing anti-alphaIFN antibodies, which are known to increase HHV8 assembly, increases C-type particle production. These data suggest that factors expressed in advanced stages of HHV8 reactivation support endogenous C-type particle morphogenesis in PEL cells.

Expression and functional activity of CXCR-4 and CCR-5 chemokine receptors in human thymocytes.

In this paper we addressed the expression of the HIV co-receptors CXCR-4 and CCR-5 in human thymocytes by phenotypic, molecular and functional approaches. Cytofluorimetric analysis disclosed that CXCR-4 was constitutively expressed by freshly isolated thymocytes (~10 000 molecules/cell in about 30% of thymocytes); the receptor was endowed with functional activity, as it mediated polarization, migration and intracellular Ca2+ increase in response to its ligand, SDF-1. On the contrary, CCR-5 expression in freshly isolated thymocytes was significantly lower (<4000 molecules/cell in less than 5% of the cells), and no functional response to CCR-5 agonists could be documented. Northern blot analysis of freshly isolated thymocytes showed high CXCR-4 mRNA levels, whereas the message for CCR-5 was barely detectable. On the other hand, a modest increase in the expression of CCR-5 was associated with in vitro thymocyte stimulation, and CCR-5 density at the cell surface attained CXCR-4 figures in most cases. None the less, no functional response to CCR-5 agonists could be documented in in vitro stimulated thymocytes. In vitro infection of thymocytes by CAT-expressing recombinant HIV bearing the envelope glycoproteins from different isolates showed that T-tropic strains, which use CXCR-4 as a co-receptor, were more efficient in infecting thymocytes than M-tropic strains, which preferentially use CCR-5. Altogether, these data indicate that expression of the major co-receptors involved in infection by M-tropic HIV strains is very poor in human thymocytes, and would suggest that thymocyte infection by M-tropic HIV strains may be a rare event in vivo.

Vaccination with DNA containing tat coding sequences and unmethylated CpG motifs protects cynomolgus monkeys upon infection with simian/human immunodeficiency virus (SHIV89.6P).

Recent evidence suggests that a CD8-mediated cytotoxic T cell response against the Tat protein of human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) controls primary infection after pathogenic virus challenge, and correlates with the status of long-term nonprogressor in humans. Due to the presence of unmethylated CpG sequences, DNA vaccination can boost the innate immunity driving more potent T cell-mediated immune responses. Therefore, cynomolgus monkeys were vaccinated with a tat-expressing vector containing defined unmethylated CpG sequences (pCV-tat). Here it is shown that the intramuscular inoculation of the pCV-tat contained primary infection with the highly pathogenic SHIV89.6P virus preventing the CD4(+) T cell decline in all the vaccinated monkeys. Undetectable virus replication and negative virus isolation correlated in all cases with the presence of anti-Tat CTLs. However, a CD8-mediated non cytolytic antiviral activity was also present in all protected animals. Of note, this activity was absent in the controls but was present in the monkey inoculated with the CpG-rich vector alone that was partially protected against viral challenge (i.e. no virus replication but positive virus isolation). These results suggest that a CTL response against Tat protects against primary infection by blocking virus replication at its early stage, in the absence of sterilizing immunity. Nevertheless, the boost of the innate immunity by CpG sequences can contribute to this protection both by driving more potent CTL responses and by inducing other CD8-mediated antiviral activities. Thus, the CpG-rich tat DNA vaccine may represent a promising candidate for preventive and therapeutic vaccination against AIDS.

SHIV89.6P pathogenicity in cynomolgus monkeys and control of viral replication and disease onset by human immunodeficiency virus type 1 Tat vaccine.

The Tat protein of human immunodeficiency virus (HIV) is produced very early after infection, plays a key role in the virus life cycle and in acquired immunodeficiency syndrome (AIDS) pathogenesis, is immunogenic and well conserved among all virus clades. Notably, a Tat-specific immune response correlates with non-progression to AIDS. Here, we show that a vaccine based on the Tat protein of HIV blocks primary infection with the simian/human immunodeficiency virus (SHIV)89.6P and prevents the CD4 T cell decline and disease onset in cynomolgus monkeys. No signs of virus replication were found in five out of seven vaccinated macaques for almost 1 year of follow-up. Since the inoculated virus (derived from rhesus or from cynomolgus macaques) is shown to be highly pathogenic in cynomolgus macaques, the results indicate efficacy of Tat vaccination in protection against highly pathogenic virus challenge. Finally, the studies of the Tat-specific immunological responses indicate a correlation of protection with a cytotoxic T cell response. Thus, a Tat-based vaccine is a promising candidate for preventive and therapeutic vaccination in humans.

CD4-independent infection of two CD4(-)/CCR5(-)/CXCR4(+) pre-T-cell lines by human and simian immunodeficiency viruses.

The infection of CD4-negative cells by variants of tissue culture-adapted human immunodeficiency virus type 1 (HIV-1) or HIV-2 strains has been shown to be mediated by the CXCR4 coreceptor. Here we show that two in vitro-established CD4(-)/CCR5(-)/CXCR4(+) human pre-T-cell lines (A3 and A5) can be productively infected by wild-type laboratory-adapted T-cell-tropic HIV-1 and HIV-2 strains in a CD4-independent, CXCR4-dependent fashion. Despite the absence of CCR5 expression, A3 and A5 cells were susceptible to infection by the simian immunodeficiency viruses SIVmac239 and SIVmac316. Thus, at least in A3 and A5 cells, one or more of the chemokine receptors can efficiently support the entry of HIV and SIV isolates in the absence of CD4. These findings suggest that to infect cells of different compartments, HIV and SIV could have evolved in vivo to bypass CD4 and to interact directly with an alternative receptor.

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.

The C-terminal half of the human immunodeficiency virus type 1 Gag precursor is sufficient for efficient particle assembly.

Human immunodeficiency virus type 1 particle assembly is directed by the Gag polyprotein Pr55(gag), the precursor for the matrix (MA), capsid (CA), and nucleocapsid proteins of the mature virion. We now show that CA sequences N terminal to the major homology region (MHR), which form a distinct domain, are dispensable for particle formation. However, slightly larger deletions which extend into the MHR severely impair particle production. Remarkably, a deletion which removed essentially all MA and CA sequences between the N-terminal myristyl anchor and the MHR reduced the yield of extracellular particles only moderately. Particle formation even exceeded wild-type levels when additional MA sequences, either from the N or the C terminus of the domain, were retained. We conclude that no distinct region between the myristyl anchor and the MHR is required for efficient particle assembly or release.

Virion-targeted viral inactivation of human immunodeficiency virus type 1 by using Vpr fusion proteins.

Inactivation of progeny virions with chimeric virion-associated proteins represents a novel therapeutic approach against human immunodeficiency virus (HIV) replication. The HIV type 1 (HIV-1) Vpr gene product, which is packaged into virions, is an attractive candidate for such a strategy. In this study, we developed Vpr-based fusion proteins that could be specifically targeted into mature HIV-1 virions to affect their structural organization and/or functional integrity. Two Vpr fusion proteins were constructed by fusing to the first 88 amino acids of HIV-1 Vpr the chloramphenicol acetyltransferase enzyme (VprCAT) or the last 18 C-terminal amino acids of the HIV-1 Vpu protein (VprIE). These Vpr fusion proteins were initially designed to quantify their efficiency of incorporation into HIV-1 virions when produced in cis from the provirus. Subsequently, CD4+ Jurkat T-cell lines constitutively expressing the VprCAT or the VprIE fusion protein were generated with retroviral vectors. Expression of the VprCAT or the VprIE fusion protein in CD4+ Jurkat T cells did not interfere with cellular viability or growth but conferred substantial resistance to HIV replication. The resistance to HIV replication was more pronounced in Jurkat-VprIE cells than in Jurkat-VprCAT cells. Moreover, the antiviral effect mediated by VprIE was dependent on an intact p6(gag) domain, indicating that the impairment of HIV-1 replication required the specific incorporation of Vpr fusion protein into virions. Gene expression, assembly, or release was not affected upon expression of these Vpr fusion proteins. Indeed, the VprIE and VprCAT fusion proteins were shown to affect the infectivity of progeny virus, since HIV virions containing the VprCAT or the VprIE fusion proteins were, respectively, 2 to 3 times and 10 to 30 times less infectious than the wild-type virus. Overall, this study demonstrated the successful transfer of resistance to HIV replication in tissue cultures by use of Vpr-based antiviral genes.

Use of murine CXCR-4 as a second receptor by some T-cell-tropic human immunodeficiency viruses.

The human CXCR-4 molecule serves as a second receptor for primary, T-cell-tropic, and laboratory-adapted human immunodeficiency virus type 1 (HIV-1) isolates. Here we show that murine CXCR-4 can support the entry of some of these HIV-1 isolates. Differences between mouse and human CXCR-4 in the ability to function as an HIV-1 receptor are determined by sequences in the second extracellular loop of the CXCR-4 protein.

Active-site residues of cyclophilin A are crucial for its incorporation into human immunodeficiency virus type 1 virions.

Human immunodeficiency virus type 1 (HIV-1) incorporates the cellular peptidyl-prolyl cis-trans isomerase cyclophilin A (CyPA), the cytosolic receptor for the immunosuppressant cyclosporin A (CsA). CsA inhibits the incorporation of CyPA and reduces HIV-1 virion infectivity but is inactive against closely related primate lentiviruses that do not interact with CyPA. The incorporation of CyPA into HIV-1 virions is mediated by a specific interaction with a proline-containing, solvent-exposed loop in the capsid (CA) domain of the Gag polyprotein. CsA, which disrupts the interaction with CA, binds at the active site of CyPA. To test whether active-site residues are also involved in the interaction with HIV-1 CA, we used a panel of previously characterized active-site mutants of human CyPA. Expression vectors for epitope-tagged wild-type and mutant CyPA were transfected into COS-gamma cells along with HIV-1 proviral DNA, and the virions produced were analyzed for the presence of tagged proteins. Cotransfection of the wild-type expression vector led to the incorporation of readily detectable amounts of epitope-tagged CyPA into HIV-1 virions. One CyPA mutant with a substantially decreased sensitivity to CsA was incorporated with wild-type efficiency, demonstrating that the requirements for binding to CsA and to HIV-1 CA are not identical. The remaining six CyPA mutants were incorporated with markedly reduced efficiency, providing in vivo evidence that HIV-1 CA interacts with the active site of CyPA.

Live attenuated simian immunodeficiency virus prevents super-infection by cloned SIVmac251 in cynomolgus monkeys.

The ability of a live attenuated simian immunodeficiency virus (SIV) to protect against challenge with cloned SIVmac251/BK28 was evaluated in four cynomolgus macaques. The intravenous infection of the C8 variant of the SIVmac251/32H virus, carrying an in-frame 12 bp deletion in the nef gene, did not affect the CD4+ and CD8+ cell counts, and a persistent infection associated with an extremely low virus burden in peripheral blood mononuclear cells (PBMCs) was established. After 40 weeks, these monkeys were challenged intravenously with a 50 MID50 dose of SIVmac251/BK28 virus grown on macaque cells. Four naive monkeys were infected as controls. Monkeys were monitored for 62 weeks following challenge. Attempts to rescue virus from either PBMCs or bone marrow from the C8-vaccinated monkeys were unsuccessful, but in two cases virus was re-isolated from lymph node cells. The presence of the SIV provirus with the C8 variant genotype maintaining its original nef deletion was shown by differential PCR in PBMCs, lymph nodes and bone marrow. Furthermore, in contrast to the control monkeys, the vaccinated monkeys showed normal levels for CD4+ and CD8+ cells, minimal lymphoid hyperplasia and no clinical signs of infection. Our results confirm that vaccination with live attenuated virus can confer protection. This appears to be dependent on the ability of the C8 variant to establish a persistent but attenuated infection which is necessary for inducing an immune response, as suggested by the persistence of a strong immune B cell memory and by the over-expression of interleukin (IL)-2, interferon-gamma and IL-15 mRNAs in PBMCs of C8-vaccinated monkeys but not in those of control monkeys.

CD4-induced interaction of primary HIV-1 gp120 glycoproteins with the chemokine receptor CCR-5.

For efficient entry into target cells, primary macrophage-tropic and laboratory-adapted human immunodeficiency viruses type 1 (HIV-1) require particular chemokine receptors, CCR-5 and CXCR-4, respectively, as well as the primary receptor CD4 (refs 1-6). Here we show that a complex of gp120, the exterior envelope glycoprotein, of macrophage-tropic primary HIV-1 and soluble CD4 interacts specifically with CCR-5 and inhibits the binding of the natural CCR-5 ligands, macrophage inflammatory protein (MIP)-1alpha and MIP-1beta (refs 7, 8). The apparent affinity of the interaction between gp120 and CCR-5 was dramatically lower in the absence of soluble CD4. Additionally, in the absence of gp120, an interaction between a two-domain CD4 fragment and CCR-5 was observed. A gp120 fragment retaining the CD4-binding site and overlapping epitopes was able to interact with CCR-5 only if the V3 loop, which can specify HIV-1 tropism and chemokine receptor choice, was also present on the molecule. Neutralizing antibodies directed against either CD4-induced or V3 epitopes on gp120 blocked the interaction of gp12O-CD4 complexes with CCR-5. These results suggest that HIV-1 attachment to CD4 creates a high-affinity binding site for CCR-5, leading to membrane fusion and virus entry.

Immunization of Macaca fascicularis with inactivated SIV preparations: challenge with human- or monkey-derived SIV and the effects of a longer immunization schedule.

In cynomolgus monkeys, we compared two human-derived SIVmac251 whole virus vaccines, a long vs short immunization schedule, and two different challenge viruses. Both vaccines induced protection after challenge with human-derived SIVmac251/32H. There was no difference between the two schedules of immunization. Seven monkeys, five of which were protected following the first challenge, were reboosted and rechallenged with monkey-derived SIVmac251, but no protection was observed. The titers of anti-human cell or -SIV neutralizing antibodies were not related to protection.

Extrachromosomal human immunodeficiency virus type 1 DNA forms in fresh peripheral blood lymphocytes and in two interleukin-2-independent T cell lines derived from peripheral blood lymphocytes of an asymptomatic seropositive subject.

Two immature T cell lines (FT1 and FT4) were established after in vitro cloning of peripheral blood lymphocytes (PBLs) from an asymptomatic human immunodeficiency virus type 1 (HIV-1) seropositive, human T cell-lymphotropic virus type 1 seronegative homosexual subject. Although derived from a limiting dilution cell cloning assay, these cell lines were not recloned for this study. Their growth was independent of exogenous interleukin-2. Both cell lines were able to form colonies when cloned in agar, but failed to form solid tumours when injected into nude mice. FT lines belong to the very immature T cell lineage as they exhibit rearranged TCR genes but no expression of T cell membrane antigens, including CD2, CD3, CD4, CD6, CD7 and CD8. They also contain an HIV-1 genome that was detected only in an extra-chromosomal DNA form, even after several passages in vitro. The presence of unintegrated viral DNA was also detected by polymerase chain reaction analysis in the same sample of fresh uncultured PBLs. Furthermore, despite the absence of CD4 expression, both T cell lines were susceptible to CD4-independent HIV-1 superinfection (lack of superinfection inhibition in the presence of OKT4A monoclonal antibodies).

Vaccination of pregnant cynomolgus monkeys with whole formalin-inactivated SIVmac251.

Five pregnant (two to three and one-half months) Macaca fascicularis seroconverted following immunization with sucrose-gradient purified and formalin-inactivated whole simian immunodeficiency virus (SIVmac251). No untoward effects on fetal maturation were observed during the immunization of the mothers. Antibodies to SIVmac251 (also those with in vitro neutralizing activity) were passively transferred to the offspring but disappeared within two to six months after birth. Antibodies to env glycoprotein (gp130) lasted longer than those against viral gag proteins (p26,p60).