Xenoinfection of nonhuman primates by feline immunodeficiency virus.

New viral infections in humans usually result from viruses that have been transmitted from other species as zoonoses. For example, it is accepted widely that human immunodeficiency virus (HIV) is the result of the propagation and adaptation of a simian immunodeficiency virus (SIV) from nonhuman primates to man [1]. Previously, we reported productive infection of primary human cells in vitro by feline immunodeficiency virus (FIV) [2], a lentivirus that causes an immunodeficiency syndrome in cats similar to HIV in humans [3]. The present study extends these findings by demonstrating that cynomolgus macaques (Macaca fasicularis) infected with FIV exhibited clinical signs, including depletion of CD4+ cells and weight loss, that are consistent with FIV infection. The development of an antibody response to FIV gag-encoded proteins and detection of virus-specific sequences in sera, blood-derived cells, and necropsied tissue accompanied these changes. Moreover, the reactivation of FIV replication from latently infected cells was observed after stimulation in vitro with phorbol esters and in vivo with tetanus toxoid. The proposed use of lentiviruses in human gene therapy [4, 5] and of nonhuman cells and organs in xenotransplantation [6] has raised concerns about zoonoses as potential sources of new human pathogens. Therefore, the study of FIV infection of primate cells may provide insight into the principles underlying retroviral xenoinfections.

Immunization as a tool to combat antimicrobial resistance.

Vaccines and immunization programs can play a key role in addressing the growing challenge of antimicrobial resistance (AMR). Amongst the high priority vaccines in development are several AMR pathogens, including: Clostridium difficile, Staphylococcus aureus, Streptococcus pneumoniae, Mycobacterium tuberculosis and Neisseria gonorrhoeae. There is evidence that vaccination can reduce the prevalence of AMR microbes, as demonstrated by both pneumococcal and Haemophilus influenza b vaccines. Research continues on many vaccine-preventable diseases, many of these AMR pathogens, including HIV and universal influenza vaccines. Not only do vaccines prevent infections, they can also prevent secondary opportunistic infections from AMR microbes-for example, bacterial pneumonia following influenza infections. The reduced need to treat these opportunistic infections would also mitigate the advance of AMR microbes in our communities. However, vaccines are not a panacea. One downside to the use of vaccines to address AMR is vaccine hesitancy, which undermines efforts to achieve herd immunity, but this is being increasingly addressed by public health education campaigns.

Attenuated SIV imparts immunity to challenge with pathogenic spleen-derived SIV but cannot prevent repair of the nef deletion.

To date, some success has been achieved with several experimental vaccines against AIDS in the available animal models. In the simian immunodeficiency virus (SIV) macaque model protection against superinfection was obtained by preinfection with a virus attenuated by a deletion in nef. To investigate the efficacy of SIVmac32H(pC8), a nef deletion mutant of SIVmac251, as a live-attenuated vaccine, rhesus monkeys were infected intravenously (i.v.) with this virus. All monkeys became productively infected by the pC8 virus. The animals had low cell-associated viral loads but developed a strong cellular and humoral antiviral immune response. Two out of eight preinfected monkeys developed signs of immunodeficiency and were excluded from the challenge. Sequence analysis of reisolates from one of them revealed a complete repair of the nef deletion. The remaining six monkeys, two preinfected for 42 weeks and four for 22 weeks, were challenged i.v. with a pathogenic SIV derived ex vivo from the spleen of a SIV infected macaque. Four of the monkeys challenged resisted the second infection whereas in two monkeys preinfected for 22 weeks full length nef was detectable. All monkeys maintained a virus-specific CD4-cell proliferative response after challenge. Thus, even after short preinfection periods with an attenuated SIV sterilising immunity against a challenge with a pathogenic SIV can be obtained. However, such a vaccine is unsafe since the attenuated virus frequently reverts to a more virulent form.

Amplicor HIV monitor, NASBA HIV-1 RNA QT and quantiplex HIV RNA version 2.0 viral load assays: a Canadian evaluation.

BACKGROUND: HIV-1 viral load quantitation is now recognized as a useful tool to monitor the efficiency of antiviral treatment and a powerful predictor of disease outcome. Three HIV-1 viral load quantitation methods have been currently available as commercial kits in Canada since 1996. OBJECTIVE: To evaluate the ability to quantify HIV-1 RNA in plasma of the Amplicor HIV Monitor Test, the NASBA HIV-1 RNA QT Assay and the Quantiplex HIV RNA Assay, version 2.0, at comparable lower detection limits. STUDY DESIGN: Blood was collected from 50 HIV-1-infected patients at various stages of infection and therapy. CD4+ cell count were estimated by flow cytometry. Plasma was isolated and tested in duplicate on four occasions using viral load kits from a single lot. HIV RNA data, performance, sensitivity and intra- and inter-assay variability were compared. RESULTS: RNA could be quantified in 33 patients by each technique. An inverse correlation was observed between viral load level and CD4+ cell counts in patients with counts below 200. Monitor could detect RNA in 94% of patients, but it showed the greatest variability and failure rate. Quantiplex 2.0 could detect HIV-1 RNA in 78%, and NASBA in 88% of the patients at theoretically equivalent lower detection limits, suggesting that the detection limit of Quantiplex 2.0 may be higher than 500 HIV-1 RNA copies per ml. NASBA had the fewest invalid tests and good reproducibility, comparable to that of Quantiplex 2.0. The mean values from NASBA and Monitor were the most similar but the best correlation was observed between Monitor and Quantiplex 2.0 results. CONCLUSIONS: Monitor, NASBA and Quantiplex results were comparable, although those obtained by Quantiplex were significantly lower. Performing this study at comparable detection limits showed that the detection limit of Quantiplex 2.0 may be higher than stated by the manufacturer.

Persistence of restricted CD4 T cell expansions in SIV-infected macaques resistant to SHIV89.6P superinfection.

Attempts to evaluate the protective effect of live attenuated SIV vaccine strains have yielded variable results depending on the route of immunization, the level of attenuation, the level of divergence between the vaccine candidate and the challenge. The protective mechanisms induced by these vaccines are still not well understood. In an effort to address whether the diversity of the CD4+ T cell repertoire in cynomolgus macaques plays a role in the immunological protection following SIVmacC8 infection, we have performed a longitudinal follow-up of the CD4 repertoire by heteroduplex tracking assay in macaques mock-infected or infected with either the attenuated SIVmacC8 or its homologous SIVmacJ5 and challenged with simian-human immunodeficiency virus (SHIV89.6P). Viral load and CD4 absolute counts were determined in these animals and the presence of SHIV89.6P virus in challenged animals was evaluated by PCR and serology. In all macaques that were protected against the challenging virus, we demonstrated a reduced diversity in the CD4+ TRBV repertoire and a few dominant CD4+ T cell clones during early primary infection. In contrast, CD4 TRBV repertoire in unprotected macaques remained highly diverse. Moreover, some of the CD4 T cell clones that were expanded during primary SIV infection re-emerged after challenge suggesting their role in protection against the challenging virus. These results underline the importance of maintaining the CD4 T cell repertoire developed during acute infection and point to the restriction of the CD4 response to the vaccine as a correlate of protection.

The 5'-terminal sequence of VSV(NJ) (Ogden): is the interaction of the NS protein with the NS binding site responsible for heterotypic interference activity?

The 5'-terminal sequence of VSV(NJ) (Ogden) and VSV(NJ) (Hazelhurst) was compared in an attempt to understand why the defective interfering particle, DI-LT, heterotypically interferes with VSV(NJ) (Ogden) but not with VSV(NJ) (Hazelhurst). The 5'-terminal sequence of VSV(NJ) (Ogden) genomic RNA was determined by direct RNA sequencing and by DNA sequencing of cDNA clones of the 3'-terminal sequence of VSV(NJ) (Ogden) DI particle genome. Primer extension analysis of the 5'-terminus of VSV(NJ) (Ogden) standard genomic RNA confirmed these data. Within the last 47 nucleotides, equivalent to the negative-strand leader RNA, the only nucleotide changes between VSV(NJ) (Ogden) and VSV(NJ) (Hazelhurst) occur between nucleotides 19 and 26, representing part of the putative NS binding region described by Isaac and Keene (J. Virol. 43, 241-249 (1982] for VSV(IND) DI particles. The spacer (S) region, located between the polyadenylation signal of the L gene and the 47th nucleotide of the leader RNA, contains more differences. The polyadenylation signal of the L gene is fully conserved, but the remainder of the L gene region (177 nucleotides) has highly diverged between VSV(NJ) (Ogden) and VSV(NJ) (Hazelhurst). The changes in the NS binding region of the negative-strand leader RNA provide further evidence for the divergent evolution of VSV(NJ) (Ogden) and VSV(NJ) (Hazelhurst). The NS binding region has been implicated as a crucial site for the initiation of RNA transcription and replication. The interaction of the NS protein with this site may determine the ability of DI particles to interfere heterotypically.

The simian immunodeficiency virus transmembrane protein is poorly immunogenic in inactivated virus vaccine.

The transmembrane proteins (TMP) of immunodeficiency lentiviruses are primary candidates for inclusion in AIDS vaccines, the design and testing of which is facilitated by the SIV-macaque infection model. Antibody responses to linear determinants in the SIVmac TMP were investigated in rhesus macaques either infected with the SIVmac J5 molecular clone or vaccinated with partially purified, formalin-inactivated SIVmac. Infected animals were shown to recognise predominantly four regions in the external domain and three regions in the internal domain of the TMP defined by a series of nominally 20mer overlapping peptides. In contrast SIV vaccinates had extremely restricted and weak antibody responses to the TMP, indicating a selective loss of immunogenicity of this component in the vaccine.

Defective interfering particles of VSVNJ (Ogden), generated by heat treatment, contain multiple internal genomic deletions.

Defective interfering (DI) particles have been isolated from a heat-resistant strain of the New Jersey (Ogden) serotype of vesicular stomatitis virus (VSV). Most of these DI particles contain various portions of all five cistrons of VSV. The two largest DI particles, NJ-121 and NJ-PG2, represent approximately 60% of the standard virus genome and contain both the positive and negative strand leader RNA templates. These two DI particles are transcriptionally active and synthesize both the positive and negative strand leader RNAs in vitro. Virion RNA probe-mRNA hybridizations and cDNA probe-virion RNA hybridizations have shown that NJ-121 contains mainly sequences from the L and G genes. In contrast, NJ-PG2 has portions of the sequences from all five genes of VSV. Smaller DI particles, NJ-121a, NJ-121b, NJ-PG1, and NJ-JM2 representing approximately 50, 38, 28, and 25% of the standard virus genome respectively, were also generated. These DI particles did not have sequences complementary to the positive strand leader RNA template. The mRNA hybridization patterns and results of the genomic RNAs hybridizing to cDNAs of N, NS, M, and G genes of these DI particles showed that they contain parts of information from all five cistrons. Most of the DI particles appear to be generated by multiple deletions throughout the standard virus genome. None of these DI particles interfered heterotypically with VSVIND-HR in BHK21, R(B77), or L2 cells. However, they interfered well with infection by VSVNJ (Hazelhurst).

Expression, characterization and purification of simian immunodeficiency virus soluble, oligomerized gp160 from mammalian cells.

The envelope glycoprotein, gp160, of human (HIV) and simian (SIV) immunodeficiency viruses mediates virus-host cell binding followed by fusion of the viral and plasma membranes. The envelope proteins are known to exist as non-covalently associated oligomers on the virus surface. The production of permanent mammalian cell lines that constitutively secrete relatively high levels of soluble forms of SIV gp160 is described and we show that these proteins are secreted predominantly as tetramers with lower levels of dimer forms. Oligomeric forms were purified to greater than 90% purity using a simple gel filtration method. The purified proteins bind CD4 suggesting that they remain in their native conformation. The purified oligomeric proteins provide the basis for more relevant structural, functional and immunological studies than recombinant gp120 as they more closely resemble the envelope protein oligomer.

Nucleotide sequence analysis of the L gene of vesicular stomatitis virus (New Jersey serotype): identification of conserved domains in L proteins of nonsegmented negative-strand RNA viruses.

We have determined the nucleotide sequence of the L gene of vesicular stomatitis virus (VSV), New Jersey serotype (Ogden strain) by primer extension dideoxy sequencing of the genomic RNA with reverse transcriptase. This analysis completes the entire genomic sequence of the VSVNJ (Ogden). Comparison of the deduced amino acid sequence of this L protein with those reported for L proteins of Indiana serotype and Hazelhurst strain of New Jersey serotype revealed an extensive sequence similarity among all three proteins. The comparison was further extended to the L proteins of other nonsegmented negative-strand RNA viruses, namely the rabies virus and four members of the paramyxovirus family: measles, Newcastle disease, human parainfluenza 3, and Sendai viruses. Our findings confirmed the existence of conserved as well as unique domains in the L proteins, suggesting an evolutionary relationship among these viruses.

Prior infection with a nonpathogenic chimeric simian-human immunodeficiency virus does not efficiently protect macaques against challenge with simian immunodeficiency virus.

Prior infection with a nef-deleted simian immunodeficiency virus (SIV) protects macaques not only against a homologous pathogenic SIV challenge but also against challenge with a chimeric SIV expressing a human immunodeficiency virus type 1 env gene (SHIV). Since this SHIV is itself nonpathogenic, we sought to explore the use of a nonpathogenic SHIV as a live, attenuated AIDS virus vaccine. Four cynomolgus monkeys infected for greater than 600 days with a chimeric virus composed of SIVmac 239 expressing the human immunodeficiency virus type 1 HXBc2 env, tat, and rev genes were challenged intravenously with 100 animal infectious doses of the J5 clone of SIVmac 32H, an isolate derived by in vivo passage of SIVmac 251. Three of the four monkeys became infected with SIVmac. This observation underlines the difficulty, even with a live virus vaccine, in protecting against an AIDS virus infection.

Repair and evolution of nef in vivo modulates simian immunodeficiency virus virulence.

Experimental evidence from the simian immunodeficiency virus (SIV) model of AIDS has shown that the nef gene is critical in the pathogenesis of AIDS. Consequently, nef is of considerable interest in both antiviral drug and vaccine development. Preliminary findings in two rhesus macaques indicated that a deletion of only 12 bp found in the overlapping nef/3' long terminal repeat (LTR) region (9501 to 9512) of the SIVmacC8 molecular clone was associated with reduced virus isolation frequency. We show that this deletion can be repaired in vivo by a sequence duplication event and that sequence evolution continues until the predicted amino acid sequence of the repair is virtually indistinguishable from that of the virulent wild type. These changes occurred concomitantly with reversion to virulence, evidenced by a high virus isolation frequency and load, decline in anti-p27 antibody, substantial reduction in the CD4/CD8 ratio, and development of opportunistic infections associated with AIDS. These findings clearly illustrate the capacity for repair of small attenuating deletions in primate lentiviruses and also strongly suggest that the region from 9501 to 9512 in the SIV nef/3' LTR region is of biological relevance. In addition, the ability of attenuated virus to revert to virulence raises fundamental questions regarding the nature of superinfection immunity.

Presence of circulating CTL induced by infection with wild-type or attenuated SIV and their correlation with protection from pathogenic SHIV challenge.

The aim of this study was to evaluate the role of CTLs in the protection from challenge with pathogenic SHIV in macaques vaccinated with attenuated virus. More specifically, we have analyzed the CTL response in cynomolgus macaques vaccinated/infected with the attenuated SIVmacC8 or the wild-type SIVmacJ5 and correlated these responses to the protection from SHIV89.6P challenge. SIVmacC8-vaccinated monkeys demonstrated a broader CTL response than the SIVmacJ5-infected animals. Nevertheless, CTL against some proteins in SIVmacC8-vaccinated monkeys became progressively more difficult to detect through the day of challenge. In regards to protection from superinfection with SHIV89.6P, neither the presence of circulating CTL nor the CTL precursor frequency against any of the tested proteins correlated with the outcome of the challenge when SIVmacJ5- and SIVmacC8-infected animals were analyzed together. By analyzing the SIVmacC8-vaccinated animals separately, only the protected animal had detectable CTL precursors with moderate frequencies against all three tested proteins at the day of challenge.

Molecular and biological characterization of simian immunodeficiency virus macaque strain 32H proviral clones containing nef size variants.

The proviral genome of the 32H reisolate of simian immunodeficiency of macaques (SIVmac32H) has been cloned and sequenced. Including both long terminal repeats, it is 10277 base pairs in length and contains open reading frames for all known SIV genes (gag, pol, vif, vpx, vpr, tat, rev, env and nef). This is the first report of an infectious SIVmac molecular clone which contains no premature termination codons. Three molecular clones of SIVmac32H have been constructed differing in sequence only within their last 1.2 kb. Two of the molecular clones, SIVmac32H(pJ5) and SIVmac32H (pC8), differ in the nef coding region by an in-frame deletion of four amino acids in pC8 and two conservative amino acid changes; other nucleotide changes in the 3' LTR were not associated with known functionally critical motifs. The third clone, SIVmac32H(pB1), contains the last 1.2 kb of the SIVmac251 clone pBK28. The biological properties of virus produced after electroporation of these clones into C8166 cells has been assessed by infection of rhesus and cynomolgus macaques, time to seroconversion and by induction of cytopathic effects upon co-cultivation of infected rhesus peripheral blood lymphocytes with C8166 cells. The viruses obtained from these clones have identical growth kinetics in vitro but differ in their ability to persist in macaques. Macaques infected with pJ5 derived virus remain viraemic longer than macaques infected with pC8-derived virus. PCR analysis of circulating provirus indicates that the nef gene evolved over time in pJ5 virus-infected macaques, whereas late in infection in pC8 virus-infected macaques the nef gene remained invariant in sequence. These results support the observation that a nef deletion mutant of SIVmac239 lost its pathogenic potential and resulted in low-level viraemia when rhesus macaques were infected. Virus challenge pools for vaccine studies have been prepared for pJ5 using both human and monkey cell substrates and these stocks have been titrated both in vitro and in vivo. Virus has also been prepared from pC8 and titrated in vitro. This virus pool is being assessed as an attenuated live-virus vaccine in macaques. Since only virus originating from the SIVmac239 molecular clone is known to cause AIDS-like symptoms in rhesus macaques consistently, the SIVmac32H molecular clones should tell us more about which viral sequence features are important for the pathogenesis of AIDS.

Vaccine-induced virus-neutralizing antibodies and cytotoxic T cells do not protect macaques from experimental infection with simian immunodeficiency virus SIVmac32H (J5).

To gain further insight into the ability of subunit vaccines to protect monkeys from experimental infection with simian immunodeficiency virus (SIV), two groups of cynomolgus macaques were immunized with either recombinant SIVmac32H-derived envelope glycoproteins (Env) incorporated into immune-stimulating complexes (iscoms) (group A) or with these SIV Env iscoms in combination with p27gag iscoms and three Nef lipopeptides (group B). Four monkeys immunized with recombinant feline immunodeficiency virus Env iscoms served as controls (group C). Animals were immunized intramuscularly at weeks 0, 4, 10, and 16. Two weeks after the last immunization, monkeys were challenged intravenously with 50 monkey 50% infectious doses of virus derived from the J5 molecular clone of SIVmac32H propagated in monkey peripheral blood mononuclear cells. High titers of SIV-neutralizing antibodies were induced in the monkeys of groups A and B. In addition, p27gag-specific antibodies were detected in the monkeys of group B. Vaccine-induced cytotoxic-T-lymphocyte precursors against Env, Gag, and Nef were detected on the day of challenge in the monkeys of group B. Env-specific cytotoxic-T-lymphocyte precursors were detected in one monkey from group A. In spite of the observed antibody and T-cell responses, none of the monkeys was protected from experimental infection. In addition, longitudinal determination of cell-associated virus loads at weeks 2, 4, 6, 9, and 12 postchallenge revealed no significant differences between vaccinated and control monkeys. These findings illustrate the need to clarify the roles of the different arms of the immune system in conferring protection against primate lentivirus infections.

Macaques infected with live attenuated SIVmac are protected against superinfection via the rectal mucosa.

Good protection against systemic challenge in the SIVmac model of AIDS has been provided by prior infection with attenuated virus. To determine if such protection extends to intrarectal mucosal challenge two molecular clones, SIVmacC8 and SIVmacJ5, were used in this study. SIVmacC8 has an attenuated phenotype in vivo, due to a 12-bp deletion in the nef/ 3'-LTR, whereas SIVmacJ5 has a full size nef open reading frame and induces AIDS in infected macaques. The J5 molecular clone was shown to infect rhesus macaques following atraumatic intrarectal inoculation. The dynamics were similar to those following intravenous inoculation resulting in early, high, cell-associated viremia and seroconversion. Four macaques previously infected with the attenuated SIVmacC8 resisted superinfection with SIVmacJ5, following intrarectal inoculation. These animals also resisted intrarectal infection with an HIV/SIV chimeric virus (SHIV) composed of SIVmac239 expressing the HXBc2 env, tat, and rev genes, suggesting that immunity to the envelope proteins was unlikely to be involved in the superinfection resistance. Infection with the attenuated SIVmac generated cytotoxic T lymphocytes (CTL) detectable in the peripheral circulation, serum neutralizing antibodies, and SIV-binding antibodies in rectal fluids. SIVmacC8 proviral DNA was found in lymph nodes removed at necropsy but there was no evidence for local sequestration of challenge virus. SIV-specific CTL, were detected in gut-associated lymph nodes and may have a role in limiting superinfection following mucosal exposure.

Rapid development of vaccine protection in macaques by live-attenuated simian immunodeficiency virus.

Convincing data on experimental vaccines against AIDS have been obtained in the simian immunodeficiency virus (SIV) macaque model by preinfection with a virus attenuated by a nef deletion. To investigate the efficacy of a nef deletion mutant of SIVmac32H called pC8 as a live-attenuated vaccine after shorter preinfection periods and to learn more about the nature of the immune protection induced, eight rhesus monkeys were infected intravenously with the pC8 virus. All monkeys became persistently infected, exhibiting low cell-associated viral loads, but strong cellular and, in terms of binding antibodies, strong humoral antiviral responses. Two of eight pC8-infected monkeys developed an immunodeficiency and were not challenged. Sequence analysis of their nef revealed complete replenishment of the deletion. The other six monkeys, two preinfected for 42 weeks and four for 22 weeks, were challenged with pathogenic spleen-derived SIV. Complete protection was achieved in four vaccinees. Virus was consistently detected in two vaccinees from the 22-week-group challenge, however, they remained clinically healthy over a prolonged period. Protection from challenge virus infection or a delayed disease development seemed to be associated with a sustained SIV-specific T helper cell response after challenge. Thus, a sterilizing immunity against superinfection with pathogenic SIV can be induced even after a relatively short waiting period of 22 weeks. Nevertheless, such a vaccine raises severe safety concerns because of its potential to revert to virulence.