Showing papers on "Viremia published in 2000"

Dengue Viremia Titer, Antibody Response Pattern, and Virus Serotype Correlate with Disease Severity

Abstract: Viremia titers in serial plasma samples from 168 children with acute dengue virus infection who were enrolled in a prospective study at 2 hospitals in Thailand were examined to determine the role of virus load in the pathogenesis of dengue hemorrhagic fever (DHF). The infecting virus serotype was identified for 165 patients (DEN-1, 46 patients; DEN-2, 47 patients; DEN-3, 47 patients, DEN-4, 25 patients). Patients with DEN-2 infections experienced more severe disease than those infected with other serotypes. Eighty-one percent of patients experienced a secondary dengue virus infection that was associated with more severe disease. Viremia titers were determined for 41 DEN-1 and 46 DEN-2 patients. Higher peak titers were associated with increased disease severity for the 31 patients with a peak titer identified (mean titer of 107.6 for those with dengue fever vs. 108.5 for patients with DHF, P=.01). Increased dengue disease severity correlated with high viremia titer, secondary dengue virus infection, and DEN-2 virus type.

Analysis of Successful Immune Responses in Persons Infected with Hepatitis C Virus

Abstract: Although hepatitis C virus (HCV) infection is very common, identification of patients during acute infection is rare. Consequently, little is known about the immune response during this critical stage of the disease. We analyzed the T lymphocyte response during and after acute resolving HCV infection in three persons, using interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) and human histocompatibility leukocyte antigen (HLA) peptide tetramer assays. Acute infection was associated with a broadly directed T helper and cytotoxic T lymphocyte (CTL) response, which persisted after resolution of clinical hepatitis and clearance of viremia. At the earliest time point studied, highly activated CTL populations were observed that temporarily failed to secrete IFN-γ, a “stunned” phenotype, from which they recovered as viremia declined. In long-term HCV-seropositive persons, CTL responses were more common in persons who had cleared viremia compared with those with persistent viremia, although the frequencies of HCV-specific CTLs were lower than those found in persons during and after resolution of acute HCV infection. These studies demonstrate a strong and persistent CTL response in resolving acute HCV infection, and provide rationale to explore immune augmentation as a therapeutic intervention in chronic HCV infection.

The decay of the latent reservoir of replication-competent HIV-1 is inversely correlated with the extent of residual viral replication during prolonged anti-retroviral therapy.

Abstract: Replication-competent HIV-1 can be isolated from infected patients despite prolonged plasma virus suppression by anti-retroviral treatment1,2,3. Recent studies have identified resting, memory CD4+ T lymphocytes as a long-lived latent reservoir of HIV-1 (refs. 4,5). Cross-sectional analyses indicate that the reservoir is rather small, between 103 and 107 cells per patient5,6. In individuals whose plasma viremia levels are well suppressed by anti-retroviral therapy, peripheral blood mononuclear cells containing replication-competent HIV-1 were found to decay with a mean half-life of approximately 6 months7, close to the decay characteristics of memory lymphocytes in humans and monkeys8,9,10. In contrast, little decay was found in a less-selective patient population11. We undertook this study to address this apparent discrepancy. Using a quantitative micro-culture assay, we demonstrate here that the latent reservoir decays with a mean half-life of 6.3 months in patients who consistently maintain plasma HIV-1 RNA levels of fewer than 50 copies/ml. Slower decay rates occur in individuals who experience intermittent episodes of plasma viremia. Our findings indicate that the persistence of the latent reservoir of HIV-1 despite prolonged treatment is due not only to its slow intrinsic decay characteristics but also to the inability of current drug regimens to completely block HIV-1 replication.

Relationship between pre-existing viral reservoirs and the re-emergence of plasma viremia after discontinuation of highly active anti-retroviral therapy.

Abstract: We examined the pathogenic significance of the latent viral reservoir in the resting CD4+ T cell compartment of HIV-1-infected individuals as well as its involvement in the rebound of plasma viremia after discontinuation of highly active anti-retroviral therapy (HAART). Using heteroduplex mobility and tracking assays, we show that the detectable pool of latently infected, resting CD4+ T cells does not account entirely for the early rebounding plasma HIV in infected individuals in whom HAART has been discontinued. In the majority of patients examined, the rebounding plasma virus was genetically distinct from both the cell-associated HIV RNA and the replication-competent virus within the detectable pool of latently infected, resting CD4 + T cells. These results indicate the existence of other persistent HIV reservoirs that could prompt rapid emergence of plasma viremia after cessation of HAART and underscore the necessity to develop therapies directed toward such populations of infected cells.

Recombinant chimeric yellow fever-dengue type 2 virus is immunogenic and protective in nonhuman primates.

Abstract: A chimeric yellow fever (YF)-dengue type 2 (dengue-2) virus (ChimeriVax-D2) was constructed using a recombinant cDNA infectious clone of a YF vaccine strain (YF 17D) as a backbone into which we inserted the premembrane (prM) and envelope (E) genes of dengue-2 virus (strain PUO-218 from a case of dengue fever in Bangkok, Thailand). The chimeric virus was recovered from the supernatant of Vero cells transfected with RNA transcripts and amplified once in these cells to yield a titer of 6.3 log10 PFU/ml. The ChimeriVax-D2 was not neurovirulent for 4-week-old outbred mice inoculated intracerebrally. This virus was evaluated in rhesus monkeys for its safety (induction of viremia) and protective efficacy (induction of anti-dengue-2 neutralizing antibodies and protection against challenge). In one experiment, groups of non-YF-immune monkeys received graded doses of ChimeriVax-D2; a control group received only the vaccine diluents. All monkeys (except the control group) developed a brief viremia and showed no signs of illness. Sixty-two days postimmunization, animals were challenged with 5.0 log10 focus forming units (FFU) of a wild-type dengue-2 virus. No viremia (<1.7 log10 FFU/ml) was detected in any vaccinated group, whereas all animals in the placebo control group developed viremia. All vaccinated monkeys developed neutralizing antibodies in a dose-dependent response. In another experiment, viremia and production of neutralizing antibodies were determined in YF-immune monkeys that received either ChimeriVax-D2 or a wild-type dengue-2 virus. Low viremia was detected in ChimeriVax-D2-inoculated monkeys, whereas all dengue-2-immunized animals became viremic. All of these animals were protected against challenge with a wild-type dengue-2 virus, whereas all YF-immune monkeys and nonimmune controls became viremic upon challenge. Genetic stability of ChimeriVax-D2 was assessed by continuous in vitro passage in VeroPM cells. The titer of ChimeriVax-D2, the attenuated phenotype for 4-week-old mice, and the sequence of the inserted prME genes were unchanged after 18 passages in Vero cells. The high replication efficiency, attenuation phenotype in mice and monkeys, immunogenicity and protective efficacy, and genomic stability of ChimeriVax-D2 justify it as a novel vaccine candidate to be evaluated in humans.

Alpha/beta interferon protects adult mice from fatal Sindbis virus infection and is an important determinant of cell and tissue tropism.

Abstract: Infection of adult 129 Sv/Ev mice with consensus Sindbis virus strain TR339 is subclinical due to an inherent restriction in early virus replication and viremic dissemination. By comparing the pathogenesis of TR339 in 129 Sv/Ev mice and alpha/beta interferon receptor null (IFN-alpha/betaR(-/-)) mice, we have assessed the contribution of IFN-alpha/beta in restricting virus replication and spread and in determining cell and tissue tropism. In adult 129 Sv/Ev mice, subcutaneous inoculation with 100 PFU of TR339 led to extremely low-level virus replication and viremia, with clearance under way by 96 h postinoculation (p.i.). In striking contrast, adult IFN-alpha/betaR(-/-) mice inoculated subcutaneously with 100 PFU of TR339 succumbed to the infection within 84 h. By 24 h p.i. a high-titer serum viremia had seeded infectious virus systemically, coincident with the systemic induction of the proinflammatory cytokines interleukin-12 (IL-12) p40, IFN-gamma, tumor necrosis factor alpha, and IL-6. Replicating virus was located in macrophage-dendritic cell (DC)-like cells at 24 h p.i. in the draining lymph node and in the splenic marginal zone. By 72 h p.i. virus replication was widespread in macrophage-DC-like cells in the spleen, liver, lung, thymus, and kidney and in fibroblast-connective tissue and periosteum, with sporadic neuroinvasion. IFN-alpha/beta-mediated restriction of TR339 infection was mimicked in vitro in peritoneal exudate cells from 129 Sv/Ev versus IFN-alpha/betaR(-/-) mice. Thus, IFN-alpha/beta protects the normal adult host from viral infection by rapidly conferring an antiviral state on otherwise permissive cell types, both locally and systemically. Ablation of the IFN-alpha/beta system alters the apparent cell and tissue tropism of the virus and renders macrophage-DC-lineage cells permissive to infection.

Efficacy of postexposure prophylaxis after intravaginal exposure of pig-tailed macaques to a human-derived retrovirus (human immunodeficiency virus type 2).

Abstract: Postexposure prophylaxis (PEP) after intravaginal exposure to human immunodeficiency virus (HIV) was investigated using the HIV type 2 (HIV-2)/pig-tailed macaque transmission model. PEP for 28 days with the reverse transcriptase inhibitor (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA; tenofovir) was initiated 12 to 72 h following HIV-2 exposure. Systemic infection was not evident in the 12- and 36-h groups, as defined by plasma viremia, cell-associated provirus, antibody responses, and lymph node virus. Breakthrough infection in the 72-h group was detected at week 16 post-virus exposure. These results demonstrate for the first time using a vaginal transmission model that early intervention after high-risk sexual exposures may prevent infection.

Duration of viremia in hepatitis A virus infection.

Abstract: The duration of viremia and time course for development of IgM antibodies were determined prospectively in natural and experimental hepatitis A virus (HAV) infection. Serial serum samples from HAV-infected men (n=13) and experimentally infected chimpanzees (n=5) were examined by nested reverse-transcriptase polymerase chain reaction analysis to detect HAV RNA and by ELISA to detect IgM antibodies to HAV. Among infected humans, HAV RNA was detected an average of 17 days before the alanine aminotransferase peak, and viremia persisted for an average of 79 days after the liver enzyme peak. The average duration of viremia was 95 days (range, 36-391 days). Results were similar in chimpanzees. In addition, HAV RNA was detected in serum of humans and chimpanzees several days before IgM antibodies to HAV were detected. These results indicate that adults with HAV infection are viremic for as long as 30 days before the onset of symptoms and that the duration of viremia may be longer than previously described.

Early Therapy of Vertical Human Immunodeficiency Virus Type 1 (HIV-1) Infection: Control of Viral Replication and Absence of Persistent HIV-1-Specific Immune Responses

Abstract: Studies of potent antiretroviral combination regimens were undertaken in young infants to evaluate the potential for long-term suppression of viral replication and to evaluate the immune consequences of such therapies. Early combination antiretroviral therapy led to a loss of plasma viremia, cultivable virus, and labile extrachromosomal replication intermediates. Despite preservation of immune function, persistent human immunodeficiency type 1 (HIV-1)-specific immune responses were not detected in most infants. The absence of detectable, persisting immune responses in most HIV-1-infected infants treated early contrasts with what is typically seen in adults who are treated early. These results are consistent with the notion that early combination antiretroviral therapy of HIV-1-infected infants allows the long-term suppression of viral replication.

3-Year suppression of HIV viremia with indinavir, zidovudine, and lamivudine

Abstract: A three-drug regimen of indinavir, zidovudine, and lamivudine suppressed viremia in two thirds of the 33 study patients for at least 3 years.

Viremia control following antiretroviral treatment and therapeutic immunization during primary SIV251 infection of macaques.

Abstract: Prolonged antiretroviral therapy (ART) is not likely to eradicate human immunodeficiency virus type I (HIV-I) infection. Here we explore the effect of therapeutic immunization in the context of ART during primary infection using the simian immunodeficiency virus (SIV251) macaque model. Vaccination of rhesus macaques with the highly attenuated poxvirus-based NYVAC-SIV vaccine expressing structural genes elicited vigorous virus-specific CD4 + and CD8+ T cell responses in macaques that responded effectively to ART. Following discontinuation of a six-month ART regimen, viral rebound occurred in most animals, but was transient in six of eight vaccinated animals. Viral rebound was also transient in four of seven mock-vaccinated control animals. These data establish the importance of antiretroviral treatment during primary infection and demonstrate that virus-specific immune responses in the infected host can be expanded by therapeutic immunization.

Genetic characterization of rebounding HIV-1 after cessation of highly active antiretroviral therapy

Abstract: Despite prolonged treatment with highly active antiretroviral therapy (HAART), infectious HIV-1 continues to replicate and to reside latently in resting memory CD4+ T lymphocytes, creating a major obstacle to HIV-1 eradication. It is therefore not surprising to observe a prompt viral rebound after discontinuation of HAART. The nature of the rebounding virus, however, remains undefined. We now report on the genetic characterization of rebounding viruses in eight patients in whom plasma viremia was undetectable throughout about 3 years of HAART. Taking advantage of the extensive length polymorphism in HIV-1 env, we found that in five patients who did not show HIV-1 replication during treatment, the rebound virus was identical to those isolated from the latent reservoir. In three other patients, two of whom had been free of plasma viremia but had showed some residual viral replication, the rebound virus was genetically different from the latent reservoir virus, corresponding instead to minor viral variants detected during the course of treatment in lymphoid tissues. We conclude that in cases with apparent complete HIV-1 suppression by HAART, viral rebound after cessation of therapy could have originated from the activation of virus from the latent reservoir. In patients with incomplete suppression by chemotherapy, however, the viral rebound is likely triggered by ongoing, low-level replication of HIV-1, perhaps occurring in lymphoid tissues.

Comparative Efficacy of Recombinant Modified Vaccinia Virus Ankara Expressing Simian Immunodeficiency Virus (SIV) Gag-Pol and/or Env in Macaques Challenged with Pathogenic SIV

Abstract: Prior studies demonstrated that immunization of macaques with simian immunodeficiency virus (SIV) Gag-Pol and Env recombinants of the attenuated poxvirus modified vaccinia virus Ankara (MVA) provided protection from high levels of viremia and AIDS following challenge with a pathogenic strain of SIV (V. M. Hirsch et al., J. Virol. 70:3741-3752, 1996). This MVA-SIV recombinant expressed relatively low levels of the Gag-Pol portion of the vaccine. To optimize protection, second-generation recombinant MVAs that expressed high levels of either Gag-Pol (MVA-gag-pol) or Env (MVA-env), alone or in combination (MVA-gag-pol-env), were generated. A cohort of 24 macaques was immunized with recombinant or nonrecombinant MVA (four groups of six animals) and was challenged with 50 times the dose at which 50% of macaques are infected with uncloned pathogenic SIVsmE660. Although all animals became infected postchallenge, plasma viremia was significantly reduced in animals that received the MVA-SIV recombinant vaccines as compared with animals that received nonrecombinant MVA (P = 0.0011 by repeated-measures analysis of variance). The differences in the degree of virus suppression achieved by the three MVA-SIV vaccines were not significant. Most importantly, the reduction in levels of viremia resulted in a significant increase in median (P < 0.05 by Student's t test) and cumulative (P = 0.010 by log rank test) survival. These results suggest that recombinant MVA has considerable potential as a vaccine vector for human AIDS.

Effect of Combination Antiretroviral Therapy on T-Cell Immunity in Acute Human Immunodeficiency Virus Type 1 Infection

Abstract: P= .01 patients. Thus, combination therapy administered within 3‐4 months of infection was associated with improved T-cell memory responses that were distinct from those of untreated patients. The amplified HIV-1‐specific T-cell responses may help maintain cytotoxic activities. The induction of cellular immunity is crucial for the successful control of many viral infections. In human immunodeficiency virus type 1 (HIV-1) infection, a vigorous cytotoxic T lymphocyte (CTL) response early in infection is associated with lower levels of plasma viremia [1‐3], and maintenance of these effector activities is correlated with clinically stable HIV1 infection [4, 5]. However, HIV-1 has evolved strategies to evade and disable the immune system [6, 7]. Thus, despite mounting HIV-specific effector activities, most untreated individuals over time sustain high levels of viremia, experience profound immunodeficiency, and eventually die of AIDS-related diseases. The ultimate failure of CTL to control HIV-1 in vivo may result largely from the inability of CD4 1 T lym

High Levels of Viral Replication during Primary Simian Immunodeficiency Virus SIVagm Infection Are Rapidly and Strongly Controlled in African Green Monkeys

Abstract: In contrast to pathogenic human immunodeficiency virus and simian immunodeficiency virus (SIV) infections, chronic SIVagm infections in African green monkeys (AGMs) are characterized by persistently low peripheral and tissue viral loads that correlate with the lack of disease observed in these animals. We report here data on the dynamics of acute SIVagm infection in AGMs that exhibit remarkable similarities with viral replication patterns observed in peripheral blood during the first 2 weeks of pathogenic SIVmac infections. Plasma viremia was evident at day 3 postinfection (p.i.) in AGMs, and rapid viral replication led by days 7 to 10 to peak viremias characterized by high levels of antigenemia (1.2 to 5 ng of p27/ml of plasma), peripheral DNA viral load (104 to 105 DNA copies/106 peripheral blood mononuclear cells [PBMC]), and plasma RNA viral load (2 × 106 to 2 × 108 RNA copies/ml). The lymph node (LN) RNA and DNA viral load patterns were similar to those in blood, with peaks observed between day 7 and day 14. These values in LNs (ranging from 3 × 105 to 3 × 106 RNA copies/106 LN cell [LNC] and 103 to 104 DNA copies/106 LNC) were at no time point higher than those observed in the blood. Both in LNs and in blood, rapid and significant decreases were observed in all infected animals after this peak of viral replication. Within 3 to 4 weeks p.i., antigenemia was no longer detectable and peripheral viral loads decreased to values similar to those characteristic of the chronic phase of infection (102 to 103 DNA copies/106 PBMC and 2 × 103 to 2 × 105 RNA copies/ml of plasma). In LNs, viral loads declined to 5 × 101 to 103 DNA copies and 104 to 3 × 105 RNA copies per 106 LNC at day 28 p.i. and continued to decrease until day 84 p.i. (<10 to 3 × 104 RNA copies/106 LNC). Despite extensive viremia during primary infection, neither follicular hyperplasia nor CD8+ cell infiltration into LN germinal centers was detected. Altogether, these results indicate that the nonpathogenic outcome of SIVagm infection in its natural host is associated with a rapidly induced control of viral replication in response to SIVagm infection, rather than with a poorly replicating virus or a constitutive host genetic resistance to virus replication.

Containment of Simian Immunodeficiency Virus Infection: Cellular Immune Responses and Protection from Rechallenge following Transient Postinoculation Antiretroviral Treatment

Abstract: To better understand the viral and host factors involved in the establishment of persistent productive infection by primate lentiviruses, we varied the time of initiation and duration of postinoculation antiretroviral treatment with tenofovir {9-[2-(R)-(phosphonomethoxy)propyl]adenine}while performing intensive virologic and immunologic monitoring in rhesus macaques, inoculated intravenously with simian immunodeficiency virus SIVsmE660. Postinoculation treatment did not block the initial infection, but we identified treatment regimens that prevented the establishment of persistent productive infection, as judged by the absence of measurable plasma viremia following drug discontinuation. While immune responses were heterogeneous, animals in which treatment resulted in prevention of persistent productive infection showed a higher frequency and higher levels of SIV-specific lymphocyte proliferative responses during the treatment period compared to control animals, despite the absence of either detectable plasma viremia or seroconversion. Animals protected from the initial establishment of persistent productive infection were also relatively or completely protected from subsequent homologous rechallenge. Even postinoculation treatment regimens that did not prevent establishment of persistent infection resulted in downmodulation of the level of plasma viremia following treatment cessation, compared to the viremia seen in untreated control animals, animals treated with regimens known to be ineffective, or the cumulative experience with the natural history of plasma viremia following infection with SIVsmE660. The results suggest that the host may be able to effectively control SIV infection if the initial exposure occurs under favorable conditions of low viral burden and in the absence of ongoing high level cytopathic infection of responding cells. These findings may be particularly important in relation to prospects for control of primate lentiviruses in the settings of both prophylactic and therapeutic vaccination for prevention of AIDS. The early stage of primary infection with the primate lentiviruses (human immunodeficiency virus (HIV) and simian immunodeficiency virus [SIV]) is an extremely dynamic period during which important aspects of the virus-host relationship are established, with far-reaching ramifications for the subsequent course of infection (18, 19, 30, 39, 51). It is a striking feature of infection with the primate lentiviruses that clearance of the initial infection is distinctly unusual. Recently it has been postulated that a major factor contributing to the failure of host containment of HIV infection is the early and continuing loss of T-cell help for development and maintenance of effective cytotoxic T-lymphocyte (CTL) responses, as a conse

Immunization with a modified vaccinia virus expressing simian immunodeficiency virus (SIV) Gag-Pol primes for an anamnestic Gag-specific cytotoxic T-lymphocyte response and is associated with reduction of viremia after SIV challenge.

Abstract: Evidence for a critical role of cytotoxic T lymphocytes (CTLs) in the containment of human immunodeficiency virus (HIV) infection (16, 23, 36, 39) has led to a consensus among those attempting to develop an AIDS vaccine that such a vaccine should generate CTL in addition to broadly neutralizing antibodies (26). An additional hurdle for an AIDS vaccine is the long-term maintenance of levels of CTL and antibody that will be necessary for protection (26). Both effector CTL and neutralizing antibodies induced by vaccination tend to be transient. Therefore, it may not be feasible to maintain immune responses essential for preventing infection. The importance of the magnitude of the vaccine-elicited memory and postinfection anamnestic immune responses thus become a critical issue in developing an AIDS vaccine. At present, viable vaccine strategies that might effectively stimulate CTL include viral vectors, peptides, and DNA immunization (26). Viral vectors under investigation include adenovirus (10, 48), alphaviruses such as Semliki Forest virus (8, 35) and Venezuelan equine encephalitis virus (11), poliovirus replicons (23), and various poxviruses (12, 21, 29, 30, 34, 43). Among these approaches, use of the poxviruses is a particularly promising vaccine strategy to express viral proteins. Studies with conventional New York Board of Health vaccinia virus demonstrated that priming with a vaccinia virus recombinant expressing simian immunodeficiency virus (SIV) envelope and/or core proteins, followed by boosting the antibody response with recombinant envelope protein, provided protection against a homologous SIV challenge with a biologically cloned strain of limited pathogenicity (SIVmne/E11S) (20). However this approach provided only partial protection against a more pathogenic and heterogeneous SIV challenge (SIVmne) (44) and blunting of viremia in macaques challenged with the highly pathogenic SIVmac251 (2). Since there are side effects associated with using conventional vaccinia viruses that become potentially life threatening when used in immunocompromised individuals (34), the use of attenuated poxviruses is an attractive alternative (20, 34, 43). A number of attenuated poxvirus strains have been developed as vaccine vectors: the avipoxviruses (43), canarypox virus, fowlpox virus, and the attenuated vaccinia virus derivatives, NYVAC (43), and modified vaccinia virus Ankara (MVA) (13, 32, 34, 43). The attenuated poxviruses appear to be safe in immunosuppressed animals (30), although their bases for attenuation differ. Thus, the avipoxviruses are genetically quite distinct from vaccinia viruses and do not complete an entire replication cycle in mammalian cells. NYVAC is a genetically engineered derivative of the Copenhagen strain with deletion of host range genes (43). MVA is a spontaneously derived attenuated variant of the Ankara strain that has multiple deletions in host range genes and genes involved in suppressing vaccinia virus-elicited immune responses (29, 30, 34). Perhaps due to these latter deletions, MVA appears to be as immunogenic as wild-type vaccinia virus strains, despite limited replication in mammalian cells (5, 9, 13, 32). In addition, MVA has an excellent safety record in humans, having been used without incident as a smallpox vaccine in approximately 100,000 individuals (30). Each of these attenuated poxvirus vectors has been evaluated in primate models and has shown some degree of efficacy (1, 4, 7, 14, 19, 22, 24, 37, 49, 50, 54). In previous studies, we explored the use of MVA as a viral vector to express SIV antigens and have evaluated its efficacy in the SIVsm-macaque model (19). The SIV-macaque model is a highly relevant system in which to evaluate the efficacy of partially protective vaccines since it provides valid disease endpoints. In addition, the level at which plasma viremia stabilizes after primary infection (or viral set point) is a highly significant prognostic surrogate for the rate of disease progression in both HIV infection (31) and SIV infection (19, 52, 55). While the SIV-HIV (SHIV) chimeras provide a system to evaluate the role of envelope-specific immune response in vaccine protection, there is no particular advantage to the use of SHIV in evaluating Gag-Pol-specific immunity. Indeed, the pathogenesis of SIV infection of macaques more closely models the pathogenesis of human AIDS (18) than the rapid CD4+ T-cell depletion observed with the pathogenic SHIVs (21, 28, 51). Prior immunization with MVA expressing the SIVsmH4 Gag-Pol and Env followed by boosting with whole inactivated SIV particles, administered without adjuvant, resulted in significant modulation of viremia and disease progression in macaques subsequently challenged with pathogenic SIVsmE660 (19). Two of these MVA-SIV vaccinees have maintained low viremia and normal CD4+ T-lymphocyte numbers throughout the 4 years since challenge, analogous to HIV type 1-infected clinical long-term nonprogressor humans (12, 42). The recent development of technology for measuring effector CTL by flow cytometric analyses of CD8+ T lymphocytes that bind specific peptide epitope-major histocompatibility complex (MHC) class I tetrameric complexes (3) has revolutionized the ability to quantitate CTL responses in SIV-infected macaques (24, 25). This technology is particularly useful for evaluation of CTL responses in recombinant vaccinia virus-immunized macaques since the background cytolysis that is seen in functional CTL assays with the use of vaccinia virus expression of viral proteins in target cells can be avoided. In a previous study we used tetramer technology to evaluate the ability of an MVA recombinant expressing SIV Gag-Pol to elicit CTL (50). In the present study, we evaluated immunogenicity of this recombinant through two subsequent boosts and evaluated the efficacy following intravenous challenge with pathogenic, uncloned, SIVsmE660.

Chimeric yellow fever virus 17D-Japanese encephalitis virus vaccine: dose-response effectiveness and extended safety testing in rhesus monkeys.

Abstract: ChimeriVax-JE is a live, attenuated recombinant virus prepared by replacing the genes encoding two structural proteins (prM and E) of yellow fever 17D virus with the corresponding genes of an attenuated strain of Japanese encephalitis virus (JE), SA14-14-2 (T. J. Chambers et al., J. Virol. 73:3095–3101, 1999). Since the prM and E proteins contain antigens conferring protective humoral and cellular immunity, the immune response to vaccination is directed principally at JE. The prM-E genome sequence of the ChimeriVax-JE in diploid fetal rhesus lung cells (FRhL, a substrate acceptable for human vaccines) was identical to that of JE SA14-14-2 vaccine and differed from sequences of virulent wild-type strains (SA14 and Nakayama) at six amino acid residues in the envelope gene (E107, E138, E176, E279, E315, and E439). ChimeriVax-JE was fully attenuated for weaned mice inoculated by the intracerebral (i.c.) route, whereas commercial yellow fever 17D vaccine (YF-Vax) caused lethal encephalitis with a 50% lethal dose of 1.67 log10 PFU. Groups of four rhesus monkeys were inoculated by the subcutaneous route with 2.0, 3.0, 4.0, and 5.0 log10 PFU of ChimeriVax-JE. All 16 monkeys developed low viremias (mean peak viremia, 1.7 to 2.1 log10 PFU/ml; mean duration, 1.8 to 2.3 days). Neutralizing antibodies appeared between days 6 and 10; by day 30, neutralizing antibody responses were similar across dose groups. Neutralizing antibody titers to the homologous (vaccine) strain were higher than to the heterologous wild-type JE strains. All immunized monkeys and sham-immunized controls were challenged i.c. on day 54 with 5.2 log10 PFU of wild-type JE. None of the immunized monkeys developed viremia or illness and had mild residual brain lesions, whereas controls developed viremia, clinical encephalitis, and severe histopathologic lesions. Immunized monkeys developed significant (≥4-fold) increases in serum and cerebrospinal fluid neutralizing antibodies after i.c. challenge. In a standardized test for neurovirulence, ChimeriVax-JE and YF-Vax were compared in groups of 10 monkeys inoculated i.c. and analyzed histopathologically on day 30. Lesion scores in brains and spinal cord were significantly higher for monkeys inoculated with YF-Vax. ChimeriVax-JE meets preclinical safety and efficacy requirements for a human vaccine; it appears safer than yellow fever 17D vaccine but has a similar profile of immunogenicity and protective efficacy.

Wide Range of Viral Load in Healthy African Green Monkeys Naturally Infected with Simian Immunodeficiency Virus

Abstract: The distribution and levels of simian immunodeficiency virus (SIV) in tissues and plasma were assessed in naturally infected African green monkeys (AGM) of the vervet subspecies (Chlorocebus pygerythrus) by limiting-dilution coculture, quantitative PCR for viral DNA and RNA, and in situ hybridization for SIV expression in tissues. A wide range of SIV RNA levels in plasma was observed among these animals (<1,000 to 800,000 copies per ml), and the levels appeared to be stable over long periods of time. The relative numbers of SIV-expressing cells in tissues of two monkeys correlated with the extent of plasma viremia. SIV expression was observed in lymphoid tissues and was not associated with immunopathology. Virus-expressing cells were observed in the lamina propria and lymphoid tissue of the gastrointestinal tract, as well as within alveolar macrophages in the lung tissue of one AGM. The range of plasma viremia in naturally infected AGM was greater than that reported in naturally infected sooty mangabeys. However, the degree of viremia in some AGM was similar to that observed during progression to AIDS in human immunodeficiency virus-infected individuals. Therefore, containment of viremia is an unlikely explanation for the lack of pathogenicity of SIVagm in its natural host species, AGM.

Large-Plaque Mutants of Sindbis Virus Show Reduced Binding to Heparan Sulfate, Heightened Viremia, and Slower Clearance from the Circulation

Abstract: Laboratory strains of Sindbis virus must bind to the negatively charged glycosaminoglycan heparan sulfate in order to efficiently infect cultured cells. During infection of mice, however, we have frequently observed the development of large-plaque viral mutants with a reduced ability to bind to heparan sulfate. Sequencing of these mutants revealed changes of positively charged amino acids in putative heparin-binding domains of the E2 glycoprotein. Recombinant viruses were constructed with these changes as single amino acid substitutions in a strain Toto 1101 background. All exhibited decreased binding to heparan sulfate and had larger plaques than Toto 1101. When injected subcutaneously into neonatal mice, large-plaque viruses produced higher-titer viremia and often caused higher mortality. Because circulating heparin-binding proteins are known to be rapidly sequestered by tissue heparan sulfate, we measured the kinetics of viral clearance following intravenous injection. Much of the parental small-plaque Toto 1101 strain of Sindbis virus was cleared from the circulation by the liver within minutes, in contrast to recombinant large-plaque viruses, which had longer circulating half-lives. These findings indicate that a decreased ability to bind to heparan sulfate allows more efficient viral production in vivo, which may in turn lead to increased mortality. Because Sindbis virus is only one of a growing number of viruses from many families which have been shown to bind to heparan sulfate, these results may be generally applicable to the pathogenesis of such viruses.

HIV-1 can be recovered from a variety of cells including peripheral blood monocytes of patients receiving highly active antiretroviral therapy: a further obstacle to eradication.

Abstract: During highly active antiretroviral therapy (HAART), HIV-1 can still persist in circulating, resting CD4+ T lymphocytes, lymph node mononuclear cells, and seminal cells of patients despite sustained suppression of plasma viremia to undetectable levels. Sanctuary sites where antiretroviral drug penetration is not optimal may allow local HIV-1 infection of cells within and passing through these tissues. Factors such as imperfect drug adherence due to complicated drug regimens may also result in tissue compartments with suboptimal drug concentrations allowing viral replication. We have examined blood monocytes from HIV-1-infected subjects being effectively treated with HAART to determine virus carriage in these cells. Monocytes were purified from peripheral blood of patients with plasma HIV-1 RNA below 50 copies/mL and who had maintained levels of plasma RNA below detection for 3 months or more. Replication-competent virus could be recovered from the majority of monocyte populations by co-culture with CD8-depleted, PHA-activated, peripheral blood mononuclear cells. Sequencing of the reverse transcriptase and protease genes of the recovered viruses did not reveal resistance to both reverse transcriptase and protease inhibitors. Continued new infection of this transitory, circulating population of cells even during prolonged, effective HAART most likely reflects ongoing, low-level HIV-1 replication within cellular reservoirs and sanctuary sites in the body.

Decay of cell-associated HIV-1 DNA correlates with residual replication in patients treated during acute HIV-1 infection.

Abstract: OBJECTIVES: To evaluate the decay rate of cell-associated HIV-1 RNA and DNA and to identify factors associated with residual viral load in patients treated at the time of primary HIV-1 infection. PATIENTS: A group of 15 patients adherent to highly active antiretroviral therapy (HAART) with sustained undetectable HIV-1 viremia for at least 24 months. METHODS: Viremia, cell-associated HIV-1 RNA and DNA in blood and lymph node mononuclear cells were measured using ultrasensitive assays. RESULTS: Viremia decreased rapidly in all patients; HIV RNA remained < 3 copies/ml in nine patients and fluctuated between 3 and 50 copies/ml in five patients and between 50 and 200 copies/ml in one patient. Decay rates of cell-associated RNA and DNA presented an inflexion point at 1 and 3 months, respectively: first-phase mean half-lives were 0.15 and 0.84 months, respectively, and second-phase mean half-lives were 13.7 and 6.6 months, respectively (95% confidence interval 4.4-13.8). The second phase decay rates were markedly slower, with a DNA decay rate that was highly associated with the mean levels of cell-associated RNA measured in blood from 6 to 33 months (P= 0.001) and in lymph nodes collected at 14 months (P= 0.02). CONCLUSIONS: The clearance of HIV-1 infected cells is correlated with the extent of viral replication as measured by cell-associated RNA levels in both blood and lymph nodes. Quantification of cell-associated RNA and DNA further defines treatment efficacy in 'aviremic' patients.

Tissue Sites of Persistent Infection and Active Replication of Equine Infectious Anemia Virus during Acute Disease and Asymptomatic Infection in Experimentally Infected Equids

Abstract: Equine infectious anemia virus (EIAV) infection of horses is characterized by recurring cycles of disease and viremia that typically progress to an inapparent infection in which clinical symptoms are absent as host immune responses maintain control of virus replication indefinitely. The dynamics of EIAV viremia and its association with disease cycles have been well characterized, but there has been to date no comprehensive quantitative analyses of the specific tissue sites of EIAV infection and replication in experimentally infected equids during acute disease episodes and during asymptomatic infections in long-term inapparent carriers. To characterize the in vivo site(s) of viral infection and replication, we developed a quantitative competitive PCR assay capable of detecting 10 copies of viral DNA and a quantitative competitive reverse transcription-PCR assay with a sensitivity of about 30 copies of viral singly spliced mRNA. Animals were experimentally infected with one of two reference viruses: the animal-passaged field isolate designated EIAVWyo and the virulent cell-adapted strain designated EIAVPV. Tissues and blood cells were isolated during the initial acute disease or from asymptomatic animals and analyzed for viral DNA and RNA levels by the respective quantitative assays. The results of these experiments demonstrated that the appearance of clinical symptoms in experimentally infected equids coincided with rapid widespread seeding of viral infection and replication in a variety of tissues. During acute disease, the predominant cellular site of viral infection and replication was the spleen, which typically accounted for over 90% of the cellular viral burden. In asymptomatic animals, viral DNA and RNA persisted in virtually all tissues tested, but at extremely low levels, a finding indicative of tight but incomplete immune control of EIAV replication. During all disease states, peripheral blood mononuclear cells (PBMC) were found to harbor less than 1% of the cellular viral burden. These quantitative studies demonstrate that tissues, rather than PBMC, constitute the predominant sites of virus replication during acute disease in infected equids and serve as resilient reservoirs of virus infection, even in the presence of highly effective immune responses that maintain a stringent control of virus replication in long-term inapparent carriers. Thus, these observations with EIAV, a predominantly macrophage-tropic lentivirus, highlight the role of tissues in sequestering lentiviral infections from host immune surveillance.

Acute sexually transmitted infections increase human immunodeficiency virus type 1 plasma viremia, increase plasma type 2 cytokines, and decrease CD4 cell counts.

Abstract: In Kenya, the median incubation time to AIDS in seroconverting sex workers is 4 years; this incubation time is specific to female sex workers. We studied the influence of acute sexually transmitted infections (STIs) on several immunologic parameters in 32 human immunodeficiency virus type 1 (HIV-1)-positive and 10 HIV-1-negative women sex workers who were followed for 1-5 months. Plasma cytokines, soluble cytokine receptors, CD4 and CD8 T cell counts, and HIV-1 plasma viremia were quantitated before, during, and after episodes of STI. Increases in interleukin (IL)-4, IL-6, IL-10, soluble tumor necrosis factor (TNF)-alpha, and viremia and a decline in CD4(+) T cell counts occurred during gonococcal cervicitis and returned to baseline after treatment. Increases in viremia correlated with increased IL-4 and decreased IL-6 concentrations. Similar changes were seen among women with acute pelvic inflammatory disease. Acute bacterial STI resulted in increased HIV-1 viremia. This may be mediated through increased inflammatory cytokines or through modulation of immune responses that control HIV-1 viremia.

Enhanced Infectivity of an R5-Tropic Simian/Human Immunodeficiency Virus Carrying Human Immunodeficiency Virus Type 1 Subtype C Envelope after Serial Passages in Pig-Tailed Macaques (Macaca nemestrina)

Abstract: Subtype C viruses have become the most prevalent human immunodeficiency virus type 1 (HIV-1) genotype globally (49). UNAIDS has estimated that there are now eight million subtype C infections worldwide, mainly in sub-Saharan Africa and Asia. In these respective geographic areas, subtype C is more common than any other subtype, and it now accounts for about 40% of all new HIV-1 infections in the world. In one recent study in two cities in southern China, 22 of 23 infected patients were found to carry subtype C viruses (Z. Chen, Y. Cao, L. Zhang, and D. Ho, unpublished data). Despite mounting efforts, it remains unclear why this subtype has gained dominance so quickly and whether means can be developed to slow down its spread. To address these questions effectively, a relevant animal model to study HIV-1 subtype C would be very useful. One of the current animal models for AIDS research consists of Asian macaques experimentally infected with simian immunodeficiency virus (SIV) (13, 14). Indeed, several molecular clones of SIV are pathogenic in vivo, causing a fatal AIDS-like disease in macaques (25, 26). For this reason, the model has been widely used to evaluate various vaccine strategies and to study AIDS pathogenesis (4, 12, 14, 19, 29, 36, 39). Nevertheless, because the env genes of SIV and HIV-1 show significant sequence diversity (28), the SIV/macaque model is of limited utility for in vivo analyses of the phenotypic and immunological properties of HIV-1 envelope. Some groups have attempted to adapt HIV-1 in macaques (2, 3, 6, 16). These efforts, however, were largely unsuccessful. The value of the macaque model has increased since the development of a chimeric simian/human immunodeficiency virus (SHIV) (31, 34, 44). Traditionally, SHIV is a chimeric lentivirus that uses pathogenic SIVmac239 as a genetic background, except that its tat, rev, and env genes are replaced by the corresponding regions of HIV-1 (23, 32, 34, 44). Since SHIV retains the ability to infect macaques, it provides a unique in vivo model for studying the pathogenic properties of HIV-1 envelope and for examining the efficacy of HIV-1 vaccines based on envelope glycoproteins. Several SHIV strains have been constructed, and their pathogenicity in nonhuman primates has been evaluated. Most current SHIV constructs utilize envelope genes derived from HIV-1 subtype B strains, either from lab-adapted, syncytium-inducing (SI), T-tropic viruses (HIV-1HXB2 and HIV-1NL43) or from primary, non-syncytium-inducing (NSI), M-tropic (HIV-1162), SI T-tropic (HIV-133), and dual-tropic (HIV89.6 and HIV-1DH12) isolates (23, 32, 34, 44). Since these chimeras retain biological properties of corresponding parental HIV-1 env, they have been used to reveal envelope-determined differences in the replication capacity of the SHIVs in vivo and in the induction of various virus-specific immune responses. These SHIV/macaque models have allowed researchers to explore the significance of HIV-1 env variation, as well as to evaluate vaccines based on HIV-1 Env antigens. In addition to SHIVs based on subtype B, one has been successfully developed for subtype E (27). However, there has been no SHIV for subtype C. In this study, approaches similar to those used for constructing subtype B and E SHIVs were adopted to make a subtype C envelope-based SHIV. We focused on primary, NSI HIV-1 subtype C viruses, as they have been demonstrated to use CCR5 for entry (52). This subtype was selected because of its emerging dominance in the epidemic, and the particular NSI, R5-tropic phenotype was selected because it represents the dominant type of HIV-1 strains transmitted sexually (54, 55). Moreover, it has been demonstrated recently that R5-tropic viruses cause distinct pathogenic effects in comparison to X4-tropic ones (20, 50). Here, we report that a replication-competent SHIVCHN19 was generated by using HIV-1 subtype C envelope in the background of SHIV33. SHIVCHN19 was found to be different from SHIV162 in that the new virus did not infect rhesus peripheral blood mononuclear cells (PBMC) despite CD8+ T-cell depletion. The virus was, however, replication competent in CD4+ T lymphocytes of pig-tailed macaques. To test its in vivo growth capacity, SHIVCHN19 was inoculated into two pig-tailed and two rhesus macaques. We found that SHIVCHN19 replicated preferentially in pig-tailed macaques. To determine whether in vivo adaptation would enhance the infectivity of SHIVCHN19, serial passages were carried out in three groups of two pig-tailed macaques each, via intravenous blood-bone marrow transfusion. In comparison to two passage 1 (P1) pig-tailed macaques, the passages were successful as shown by (i) the increasingly elevated levels of plasma viremia in animals from later passages, (ii) the shortened doubling time of plasma virus during acute infection with each passage, (iii) faster seroconversion in P2 to P4 animals, (iv) higher levels of sustained viral load in animals from later passages, (v) the enhanced viral infectivity in rhesus PBMC, and (vi) profound CD4+ T-cell depletion in the jejunal lamina propria of P4 animals. Importantly, the serial passages did not change the viral phenotype as determined by the persistence of the R5 tropism of SHIVCHN19 isolated from the two P4 animals. Our data indicate the establishment of the first R5-tropic SHIV/macaque model for HIV-1 subtype C vaccine and pathogenesis studies.