Joanne Higgins

Viral Sanctuaries during Highly Active Antiretroviral Therapy in a Nonhuman Primate Model for AIDS

ABSTRACT Highly active antiretroviral therapy (HAART) enables long-term suppression of plasma HIV-1 loads in infected persons, but low-level virus persists and rebounds following cessation of therapy. During HAART, this virus resides in latently infected cells, such as resting CD4+ T cells, and in other cell types that may support residual virus replication. Therapeutic eradication will require elimination of virus from all reservoirs. We report here a comprehensive analysis of these reservoirs in fluids, cells, and tissues in a rhesus macaque model that mimics HAART in HIV-infected humans. This nonhuman primate model uses RT-SHIV, a chimera of simian immunodeficiency virus containing the HIV-1 reverse transcriptase (RT). Methods were developed for extraction, preamplification, and real-time PCR analyses of viral DNA (vDNA) and viral RNA (vRNA) in tissues from RT-SHIV-infected macaques. These methods were used to identify viral reservoirs in RT-SHIV-infected macaques treated with a potent HAART regimen consisting of efavirenz, emtricitabine, and tenofovir. Plasma virus loads at necropsy ranged from 11 to 28 copies of vRNA per ml. Viral RNA and DNA were detected during HAART, in tissues from numerous anatomical locations. Additional analysis provided evidence for full-length viral RNA in tissues of animals with virus suppressed by HAART. The highest levels of vDNA and vRNA in HAART-treated macaques were in lymphoid tissues, particularly the spleen, lymph nodes, and gastrointestinal tract tissues. This study is the first comprehensive analysis of the tissue and organ distribution of a primate AIDS virus during HAART. These data demonstrate widespread persistence of residual virus in tissues during HAART.

Real-time TaqMan PCR as a specific and more sensitive alternative to the branched-chain DNA assay for quantitation of simian immunodeficiency virus RNA

We developed a rapid and highly reproducible assay based on real-time PCR (TaqMan, Applied Biosystems, Foster City, CA) to quantitate simian immunodeficiency virus (SIV) RNA in plasma samples. This assay was compared with the current branched-chain DNA assay (Bayer, Emeryville, CA). Results obtained with the real-time TaqMan PCR assay were comparable to those obtained with the branched-chain DNA assay in overlapping ranges of sensitivities (r = 0.9429, p < 0.05). However, the real-time TaqMan PCR assay was capable of detecting as few as 50 copies of RNA/ml, whereas branched-chain DNA was only sensitive to 1,500 copies of RNA/ml. Therefore, several animals that tested negative by branched-chain DNA were positive by realtime TaqMan PCR. Two false positive tests were also recorded for the branched-chain DNA test. False negative and positive tests were confirmed by cell culture isolation and conventional nested RT-PCR. The SIV TaqMan assay detected a wide range of wild-type, cloned, and recombinant SIV strains with similar amplification efficiency, including SIVmac251, SIVmac239, SIVmac239 containing the 184V mutation in RT, SIV1A11, SIVmac239 delta3, SIVmac-M4, and chimeras (SHIVs) containing specific HIV-1 genes, such as reverse transcriptase (RT-SHIV) or Env (SHIV-E). In conclusion, the high sensitivity, increased specificity, wide dynamic range, simplicity, and reproducibility of the real-time SIV RNA quantitation allow the screening of large numbers of samples and make this method especially suitable for measuring both viral DNA and RNA levels during vaccine and therapy studies.

Virulence differences between two field isolates of feline immunodeficiency virus (FIV-APetaluma and FIV-CPGammar) in young adult specific pathogen free cats

The goal of this study was to identify a strain of feline immunodeficiency virus (FIV) that would be more virulent for adult cats than the prototype FIV-APetaluma and, thereby, enhance the FIV infection model for HIV-1 related research. Diehl et al. reported that one clade C strain of FIV, FIV-CPGammar, was more virulent than other known FIV isolates. Mortalities from 58 to 100% were reported for kittens 12 weeks of age and less following intravenous inoculation. A more variable and somewhat less virulent disease course was observed in neonatal to 8-10-week-old kittens infected orally, intravaginally or intrarectally with this same isolate (Obert and Hoover, 2000). However, no studies have been done with FIV-CPGammar in adult cats. Therefore, the virulence of FIV-CPGammar for young adult cats was compared to that of FIV-APetalulma, the original FIV isolate. One group of five cats were inoculated intraperitoneally with 470 TCID(50) of FIV-CPGammar in the form of pooled plasma from acutely infected cats, while a second group was infected with plasma containing the 750 TCID50 of FIV-APetaluma. The cats were observed for 20 weeks for gross signs of disease, hematologic abnormalities, time of antibody appearance, and plasma and peripheral blood mononuclear cell (PBMC) associated virus levels. Viral RNA and proviral DNA were measured by a real-time PCR, sensitive to 50 copies per milliliter. The only outward sign of disease was lymphadenopathy, which occurred at a similar time and intensity in both groups of cats. Cats infected with FIV-CPGammar were more likely to be neutropenic and lymphopenic during the first 10-12 weeks of infection than cats infected with FIV-APetaluma. Both groups of cats showed similar overall declines in absolute mean CD4 cell counts and identical concomitant increases in CD8 cells. CD4/CD8 cell ratios were also similar. Antibody, as measured by an ELISA against recombinant FIV-TM antigen, appeared in all cats by 4 weeks post-infection. The most significant differences were in plasma viral RNA and PBMC proviral DNA levels. Cats infected with FIV-CPGammar had up to 100 times higher mean levels of viral RNA during the first few weeks of infection than cats infected with FIV-APetaluma. This difference was also mirrored in levels of proviral DNA in PBMC, which were significantly higher in the FIV-CPGammar infected cats. Plasma viral RNA and PBMC proviral DNA levels were virtually identical in both groups of cats at 20 weeks post-infection. However, proviral DNA in tissues such as thymus and popliteal lymph nodes was 10-fold or so higher in FIV-CPGammar infected cats at 20 weeks and histopathologic lesions were more severe. Based on these various parameters, we concluded that FIV-CPGammar was more virulent than FIV-APetaluma in young adult cats during the 20-week study period. However, we were not able to recreate the severe and rapidly progressive disease previously reported for kittens, suggesting an age-related resistance similar to that observed previously for FIV-APetaluma (George et al., 1993).

Suppression of Virus Load by Highly Active Antiretroviral Therapy in Rhesus Macaques Infected with a Recombinant Simian Immunodeficiency Virus Containing Reverse Transcriptase from Human Immunodeficiency Virus Type 1

ABSTRACT We have modeled highly active antiretroviral therapy (HAART) for AIDS in rhesus macaques infected with a chimera (RT-SHIV) of simian immunodeficiency virus containing reverse transcriptase from human immunodeficiency virus type-1 (HIV-1). Seven RT-SHIV-infected macaques were treated with a combination of efavirenz (200 mg orally once daily), lamivudine (8 mg/kg subcutaneously once daily), and tenofovir (30 mg/kg subcutaneously once daily). Plasma viral RNA levels in all animals were reduced by more than 1,000-fold after 4 weeks and, in six of the seven animals, were reduced to undetectable levels after 10 weeks. Virus loads increased slightly between 12 and 16 weeks of treatment, associated with problems with the administration of efavirenz. After a change in the method of efavirenz administration, virus loads declined again and remained undetectable in the majority of animals for the duration of therapy. Treatment was stopped for three animals after 36 weeks of therapy, and virus loads increased rapidly. Posttreatment RT-SHIV isolates had no mutations associated with resistance to any of the three drugs. Efavirenz treatment was stopped, but lamivudine and tenofovir treatment for two other macaques was continued. The virus load in one of these two animals rebounded; virus from this animal was initially free of drug-resistance mutations but acquired the K65R mutation in reverse transcriptase at 11 weeks after efavirenz treatment was withdrawn. These results mimic HAART of HIV-1-infected humans. The RT-SHIV/rhesus macaque model should be useful for studies of tissue reservoirs and sites of residual replication that are not possible or practical with humans.

Virulence and Reduced Fitness of Simian Immunodeficiency Virus with the M184V Mutation in Reverse Transcriptase

ABSTRACT Drug-resistant mutants with a methionine-to-valine substitution at position 184 of reverse transcriptase (M184V) emerged within 5 weeks of initiation of therapy in four newborn macaques infected with simian immunodeficiency virus (SIVmac251) and treated with lamivudine (3TC) or emtricitabine [(−)-FTC] (two animals per drug). Thus, this animal model mimics the rapid emergence of M184V mutants of HIV-1 during 3TC therapy of human patients. One animal of each treatment group developed fatal immunodeficiency at 12 weeks of age, which is similar to the rapid disease course seen in most untreated SIVmac251-infected infant macaques. To further evaluate the effect of the M184V mutation on viral fitness and virulence, groups of juvenile macaques were inoculated with the molecular clone SIVmac239 with either the wild-type sequence (group A [n = 5]) or the M184V sequence (SIVmac239-184V; group B [n = 5] and group C [n = 2]). The two SIVmac239-184V-infected animals of group C did not receive any drug treatment, and in both animals the virus population reverted to predominantly wild type (184M) by 8 weeks after inoculation. The other five SIVmac239-184V-infected animals (group B) were treated with (−)-FTC to prevent reversion. Although virus levels 1 week after inoculation were lower in the SIVmac239-184V-infected macaques than in the SIVmac239-infected animals, no significant differences were observed from week 2 onwards. Two animals in each group developed AIDS and were euthanized, while all other animals were clinically stable at 46 weeks of infection. These data demonstrate that the M184V mutation in SIV conferred a slightly reduced fitness but did not affect disease outcome.

Detection of antibodies to caprine arthritis-encephalitis virus using recombinant gag proteins.

SummaryThe coding sequences of the core proteins p17 and p28 of caprine arthritis-encephalitis virus (CAEV) were amplified using the polymerase chain reaction and cloned into the plasmid expression vector p-GEX-2T. Both p17 and p28 were expressed as fusion proteins with glutathione S-transferase. The recombinant proteins were affinity purified from induced bacterial lysates using glutathione-agarose beads. The purified proteins were used in an enzyme-linked immunosorbent assay (ELISA) to detect antibodies against CAEV in goat sera and milk samples. Three different ELISA tests were developed based on p17, p28 or the combination of these two recombinant proteins (p17+p28). A comparison was made to an ELISA based on purified whole virus particles and to agar immunodiffusion test (AGID). Sera with conflicting results in the different ELISA tests were examined by Western blotting. There was a high correlation between the ELISA tests based on p17+p28 recombinant proteins and whole virus ELISA, with an estimated κ value of 0.92. Only 72–75% of the sera that tested positive in these two ELISA tests were positive in AGID. Antibodies to CAEV were detected in significantly more animals when serum samples were tested compared to milk samples. Based on the time and materials required to prepare the reagents, the recombinant based ELISA test was less expensive than the whole virus ELISA.

An experimental study of primary feline immunodeficiency virus infection in cats and a historical comparison to acute simian and human immunodeficiency virus diseases

Sixteen adolescent specific pathogen free cats were inoculated with the Petaluma strain of feline immunodeficiency virus (FIV) and two cats were then necropsied at each of 5, 10, 21, 28, 42, 56, 70, and 84 day time points following infection. Lymphadenopathy gradually increased starting at Day 10 and persisted for the duration. Gross clinical signs of fever, mild to severe malaise, anorexia, diarrhea, dehydration, and generalized soreness appeared around Day 42, peaked at Day 56, and disappeared by Days 70-84 post-infection. Leukopenia, associated initially with a mild lymphopenia and later by both a mild lymphopenia and a severe neutropenia, appeared 14-28 days following infection, troughed at Day 56, and persisted thereafter. The CD4+:CD8+ T cell ratio started to decrease around Day 28, reaching a nadir at Days 56-70. This decrease was due to a decline in the absolute numbers and percentage of CD4+ T cells and an increase in the percentage of CD8+ T cells. Significant histopathologic lesions included myeloid hyperplasia between Days 56-70 post-infection; thymitis with cortical involution and follicular hyperplasia starting at Day 42; lymphoid hyperplasia of peripheral and mesenteric nodes, spleen and tonsils beginning around Day 42; typhlitis most evident from Day 56 onward, and an interstitial nephritis and pneumonitis that was most intense after Day 42. Virus was isolated from peripheral blood mononuclear cells (PBMC) beginning 2 weeks post-infection, and plasma viremia appeared 1 week later. Plasma and PBMC-associated viremia peaked at 42-56 days following infection and decreased abruptly thereafter. Proviral DNA was detectable as early as 5 days after infection in blood leukocytes and after 10 days in other organs. The central nervous system, lungs, thymus, tonsils and mesenteric lymph nodes were the earliest sites of virus localization. Antibodies to the FIV capsid protein appeared 14 days following infection and reached peak levels by Days 42-56. Abnormalities occurring during the primary stage of FIV infection were consistent with those described for acute simian and human immunodeficiency virus-induced disease.

Efavirenz Therapy in Rhesus Macaques Infected with a Chimera of Simian Immunodeficiency Virus Containing Reverse Transcriptase from Human Immunodeficiency Virus Type 1

ABSTRACT The specificity of nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) for the RT of human immunodeficiency virus type 1 (HIV-1) has prevented the use of simian immunodeficiency virus (SIV) in the study of NNRTIs and NNRTI-based highly active antiretroviral therapy. However, a SIV-HIV-1 chimera (RT-SHIV), in which the RT from SIVmac239 was replaced with the RT-encoding region from HIV-1, is susceptible to NNRTIs and is infectious to rhesus macaques. We have evaluated the antiviral activity of efavirenz against RT-SHIV and the emergence of efavirenz-resistant mutants in vitro and in vivo. RT-SHIV was susceptible to efavirenz with a mean effective concentration of 5.9 ± 4.5 nM, and RT-SHIV variants selected with efavirenz in cell culture displayed 600-fold-reduced susceptibility. The efavirenz-resistant mutants of RT-SHIV had mutations in RT similar to those of HIV-1 variants that were selected under similar conditions. Efavirenz monotherapy of RT-SHIV-infected macaques produced a 1.82-log-unit decrease in plasma viral-RNA levels after 1 week. The virus load rebounded within 3 weeks in one treated animal and more slowly in a second animal. Virus isolated from these two animals contained the K103N and Y188C or Y188L mutations. The RT-SHIV-rhesus macaque model may prove useful for studies of antiretroviral drug combinations that include efavirenz.

Reversion of the M184V mutation in simian immunodeficiency virus reverse transcriptase is selected by tenofovir, even in the presence of lamivudine.

ABSTRACT The methionine-to-valine mutation in codon 184 (M184V) in reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) or simian immunodeficiency virus (SIV) confers resistance to (−)-2′-deoxy-3′-thiacytidine (3TC; lamivudine) and increased sensitivity to 9-[2-(phosphonomethoxy)propyl]adenine (PMPA; tenofovir). We have used the SIV model to evaluate the effect of the M184V mutation on the emergence of resistance to the combination of 3TC plus PMPA. A site-directed mutant of SIVmac239 containing M184V (SIVmac239-184V) was used to select for resistance to both 3TC and PMPA by serial passage in the presence of increasing concentrations of both drugs. Under these selection conditions, the M184V mutation reverted in the majority of the selections. Variants resistant to both drugs were found to have the lysine-to-arginine mutation at codon 65 (K65R), which has previously been associated with resistance to PMPA in both SIV and HIV. Similarly, in rhesus macaques infected with SIVmac239-184V for 46 weeks and treated daily with (−)-2′-deoxy-5-fluoro-3′-thiacytidine [(−)-FTC], there was no reversion of M184V, but this mutation reverted to 184 M in all three animals within 24 weeks of treatment with (−)-FTC and PMPA. Although the addition of PMPA to the (−)-FTC therapy induced a decrease in virus loads in plasma, these loads eventually returned to pre-PMPA levels in each case. All animals receiving this combination developed the K65R mutation. These results demonstrate that the combination of PMPA with 3TC or (−)-FTC selects for the K65R mutation and against the M184V mutation in SIV RT.

Effect of chronic feline immunodeficiency virus infection on experimental feline calicivirus-induced disease

Acute feline calicivirus (FCV) infection caused a more severe disease in chronically feline immunodeficiency virus (FIV) infected than in non-FIV infected cats. FIV infected cats shed significantly higher amounts of FCV through their saliva after FCV challenge than the non-FIV infected cats. However, there was no difference in the duration of FCV shedding. None of the cats exposed to FCV developed chronic FCV carriage. Both groups of cats mounted similar titers of neutralizing antibodies to FCV. Although FIV infected cats started out with significantly lower total lymphocyte and neutrophil numbers than the non-FIV infected cats, the transient lymphopenia and neutrophilia attributable to the FCV infection was of similar intensity in both groups of animals. There was no evidence that the underlying FIV-related disease or viremia was influenced by acute FCV infection. Acute FCV infection did not significantly alter the CD4+/CD8+ T lymphocyte ratio in FIV infected compared to non-FIV infected cats. The ongoing humoral IgG response to FIV was not affected by the FCV infection. There was no significant change in the proportion of FIV infected peripheral blood mononuclear cells during 8 subsequent weeks after FCV challenge as determined by polymerase chain reaction.