Interleukin-12 (IL-12) enhancement of the cellular immune response against human immunodeficiency virus type 1 env antigen in a DNA prime/vaccinia virus boost vaccine regimen is time and dose dependent: suppressive effects of IL-12 boost are mediated by nitric oxide

We previously demonstrated that codelivery of interleukin-12 (IL-12) with the human immunodeficiency virus type 1 (HIV-1) Env antigen from a recombinant vaccinia virus (rVV) can enhance the specific anti-Env cell-mediated immune (CMI) response. In the present study, we have investigated the effects of IL-12 in mice when it is expressed in a DNA prime/VV boost vaccine regimen. The delivery of IL-12 and Env product during priming with a DNA vector, followed by a booster with VV expressing the Env gene (rVVenv), was found to trigger the optimal CMI response compared with other immunization schedules studied. Significantly, if IL-12 is also delivered as a booster from the viral vector, an impairment of the effects of IL-12 was observed involving nitric oxide (NO), since it was overcome by specific inhibitors of inducible NO synthase. NO caused transient immunosuppression rather than impairment of viral replication. Moreover, at certain viral doses, coadministration of the NO inhibitor during the booster resulted in IL-12-mediated enhancement of the specific CD8(+) T-cell response. In addition, the dose of the IL-12-encoding plasmid (pIL-12) and the route of administration of both vectors were relevant factors for optimal CMI responses. Maximal numbers of Env-specific CD8(+) gamma interferon-secreting cells were obtained when 50 microg of pIL-12 was administered intramuscularly at priming, followed by an intravenous rVVenv boost. Our results demonstrate, in a murine model, critical parameters affecting the success of vaccination schedules based on a combination of DNA and VV vectors in conjunction with immunomodulators.     

Towards a new generation of vaccines: the cytokine IL-12 as an adjuvant to enhance cellular immune responses to pathogens during prime-booster vaccination regimens

A main goal of the industrialized world is the development of effective vaccines to control infectious diseases with major health and socio-economic impact. Current understanding of the immune response triggered during infection with pathogens causing malaria, hepatitis C and AIDS emphasizes the importance of cytotoxic T lymphocytes (CTLs) in combating these infections. This has led to the development of new vaccination strategies, some of which are in phase I/II clinical trials. Promising strategies of vaccination are based on highly attenuated viral vectors, such as Vaccinia virus (VV) in combination with heterologous like vectors naked DNA, referred to as priming/booster vaccination. While these immunization schedules increased the production of specific CTLs, there is a need to further expand the CD8+T cell population to control an infection. Among molecules that play a significant role in the modulation of the CTL response is the cytokine IL-12. Immunoregulation by IL-12 is of central importance in cell-mediated immunity (CMI) against those pathogens and tumors that are controlled by cell-mediated mechanisms, supported by Thl cells. The use of this cytokine in combination with highly immunogenic VV-derived vectors is a promising system for development of future vaccination schedules. In this review, we summarize recent data on the use of IL-12 in vaccination procedures, as well as undesired side-effects of the cytokine that can be overcome by accurate use of dose, route and time-window administration of IL-12 encoding vectors. Results described here indicate that VV IL-12-mediated enhancement of the specific CMI response against a model antigen HIV-1 env was time- and dose-dependent and that the antigen and the cytokine could be expresed from two different rVVs modulating the doses of the vectors and allowing for enhancement of a specific CMI response. Moreover, the use of IL-12 during DNA prime/VV boost regimens enhanced the specific anti-HIV-1 env cellular response 20 times compared to that generated after a single rVVenv inoculation. Variables such as: a) dose of the cytokine applied, b) time of its administration and c) routes of inoculation play a critical role in the final outcome of the response. The findings presented here can be extended to other antigens, suggesting that immunomodulatory cytokines can be useful in the development of the future vaccines against numerous infectious diseases and tumors.     

Prime-boost immunization schedules based on influenza virus and vaccinia virus vectors potentiate cellular immune responses against human immunodeficiency virus Env protein systemically and in the genitorectal draining lymph nodes

Vaccines that elicit systemic and mucosal immune responses should be the choice to control human immunodeficiency virus (HIV) infections. We have previously shown that prime-boost immunizations with influenza virus Env and vaccinia virus (VV) WR Env recombinants induced an enhanced systemic CD8(+) T-cell response against HIV-1 Env antigen. In this report, we analyzed in BALB/c mice after priming with influenza virus Env the ability of two VV recombinants expressing HIV-1 Env B (VV WR Env and the highly attenuated modified VV Ankara [MVA] Env) to boost cellular immune responses in the spleen and in the lymph nodes draining the genital and rectal tracts. Groups of mice were primed by the intranasal route with 10(4) PFU of influenza virus Env and boosted 14 days later by the intraperitoneal or intranasal route with 10(7) PFU of MVA Env or VV WR Env, while the control group received two immunizations with influenza virus Env. We found that the combined immunization (Flu/VV) increased more than 60 times the number of gamma interferon-specific CD8(+) T cells compared to the Flu/Flu scheme. Significantly, boosting with MVA Env by the intraperitoneal route induced a response 1.25 or 2.5 times (spleen or genital lymph nodes) higher with respect to that found after the boost with VV WR Env. Mice with an enhanced CD8(+) T-cell response also had an increased Th1/Th2 ratio, evaluated by the cytokine pattern secreted following in vitro restimulation with gp160 protein and by the specific immunoglobulin G2a (IgG2a)/IgG1 ratio in serum. By the intranasal route recombinant WR Env booster gave a more efficient immune response (10 and 1.3 times in spleen and genital lymph nodes, respectively) than recombinant MVA Env. However, the scheme influenza virus Env/MVA Env increased four times the response in the spleen, giving a low but significant response in the genital lymph nodes compared with a single intranasal immunization with MVA Env. These results demonstrate that the combination Flu/MVA in prime-booster immunization regimens is an effective vaccination approach to generate cellular immune responses to HIV antigens at sites critical for protective responses.     

Protection of sheep against bovine leukemia virus (BLV) infection by vaccination with recombinant vaccinia viruses expressing BLV envelope glycoproteins: correlation of protection with CD4 T-cell response to gp51 peptide 51-70.

We have previously constructed vaccinia virus (VV) recombinants containing a complete or truncated envelope (env) gene of bovine leukemia virus (BLV). Only recombinants carrying the complete env gene (VV-BLV2 and VV-BLV3) expressed env glycoprotein on the surface of virus-infected cells and produced an antibody response in rabbits. In the present study, these VV recombinants were used to immunize sheep prior to challenge with BLV-infected peripheral blood mononuclear cells. Both humoral and cell-mediated immunity were monitored in infected animals. Sheep inoculated with recombinants containing the complete env gene showed a CD4 response to a defined epitope of gp51, but this response was absent 4 months postchallenge. Anti-gp51 antibodies appeared in animals inoculated with complete env 2 weeks after challenge, reached a peak at 4 weeks, and subsequently declined over 16 months. No CD4 response was recorded in animals inoculated with recombinants containing truncated env gene (VV-BLV1). BLV-infected control animals and those animals receiving VV-BLV1 were slower to develop antibodies postchallenge, and the titers of anti-gp51 antibodies continued to increase over 16 months. Proviral DNA was detected by the polymerase chain reaction in the four groups at 6 weeks after challenge. However, it could not be detected 4 months postinfection in the VV groups inoculated with complete env. Provirus was present in the VV-BLV1 and control groups over the 16-month trial period. These results demonstrate that vaccination with VV recombinants containing the complete env gene of BLV protects sheep against infection and that protection correlated with a CD4 T-cell response to a defined epitope.     

Recombinant Vaccinia Virus Coexpressing the F Protein of Respiratory Syncytial Virus (RSV) and Interleukin-4 (IL-4) Does Not Inhibit the Development of RSV-Specific Memory Cytotoxic T Lymphocytes, whereas Priming Is Diminished in the Presence of High Levels of IL-2 or Gamma Interferon

In order to investigate if immune responses to the fusion (F) protein of respiratory syncytial virus (RSV) could be influenced by cytokines, recombinant vaccinia viruses (rVV) carrying both the F gene of RSV and the gene for murine interleukin-2 (IL-2), IL-4, or gamma interferon (IFN-γ) were constructed. In vitro characterization of rVV revealed that insertion of the cytokine gene into the VP37 locus of the vaccinia virus genome resulted in 100- to 1,000-fold higher expression than insertion of the same gene into the thymidine kinase (TK) locus. In comparison, only a two- to fivefold difference in the level of expression of the F protein was observed when the gene was inserted into either of these two loci. Mice vaccinated with rVV expressing the F protein and high levels of IL-2 or IFN-γ cleared rVV more rapidly than mice inoculated with a control rVV and developed only low levels of RSV-specific serum antibody. In addition, these recombinants were much less effective at priming RSV-specific memory cytotoxic T lymphocytes (CTL) and IFN-γ production by spleen cells than rVV expressing the F protein alone. In contrast, mice vaccinated with rVV expressing high levels of IL-4 showed signs of delayed rVV clearance. RSV-specific serum antibody responses were biased in favor of immunoglobulin G1 (IgG1) in these mice, as there was a significant reduction in IgG2a antibody responses compared with serum antibody responses in mice vaccinated with rVV expressing the F protein alone. However, vaccination with rVV expressing the F protein together with high levels of IL-4 did not alter the development of RSV-specific memory CTL or IFN-γ production by RSV-restimulated splenocytes.     

Antigenicity and immunogenicity of a synthetic human immunodeficiency virus type 1 group m consensus envelope glycoprotein

Genetic variation of human immunodeficiency virus (HIV-1) represents a major obstacle for AIDS vaccine development. To decrease the genetic distances between candidate immunogens and field virus strains, we have designed and synthesized an artificial group M consensus env gene (CON6 gene) to be equidistant from contemporary HIV-1 subtypes and recombinants. This novel envelope gene expresses a glycoprotein that binds soluble CD4, utilizes CCR5 but not CXCR4 as a coreceptor, and mediates HIV-1 entry. Key linear, conformational, and glycan-dependent monoclonal antibody epitopes are preserved in CON6, and the glycoprotein is recognized equally well by sera from individuals infected with different HIV-1 subtypes. When used as a DNA vaccine followed by a recombinant vaccinia virus boost in BALB/c mice, CON6 env gp120 and gp140CF elicited gamma interferon-producing T-cell responses that recognized epitopes within overlapping peptide pools from three HIV-1 Env proteins, CON6, MN (subtype B), and Chn19 (subtype C). Sera from guinea pigs immunized with recombinant CON6 Env gp120 and gp140CF glycoproteins weakly neutralized selected HIV-1 primary isolates. Thus, the computer-generated "consensus" env genes are capable of expressing envelope glycoproteins that retain the structural, functional, and immunogenic properties of wild-type HIV-1 envelopes.     

Protection of rhesus monkeys against infection with minimally pathogenic simian-human immunodeficiency virus: correlations with neutralizing antibodies and cytotoxic T cells

We studied the capacity of active immunization of rhesus monkeys with HIV-1 envelope protein (Env) to induce primary virus cross-reactive neutralizing antibodies to prevent infection following intravenous challenge with simian-human immunodeficiency virus (SHIV). Monkeys were immunized with the human immunodeficiency type 1 (HIV-1) strain R2 Env. Initially, the Env was expressed in vivo by an alphavirus replicon particle system, and then it was administered as soluble oligomeric gp140. Concurrently, groups of monkeys received expression vectors that encoded either simian immunodeficiency virus (SIV) gag/pol genes or no SIV genes in vivo to test the additional protective benefit of concurrent induction of virus-specific cell-mediated immune (CMI) responses. Groups of control monkeys received either the gag/pol regimen or sham immunizations. The antibodies induced by the Env immunization regimen neutralized diverse primary HIV-1 strains. Similarly, potent CMI responses were induced by the gag/pol regimen, as measured by gamma interferon enzyme-linked immunospot assays. Differences in the responses among groups of monkeys strongly suggested that there was interference between the Env and gag/pol immunization regimens. Complete protection of some of the monkeys against infection after intravenous challenge with the partially pathogenic SHIV(DH12R (Clone 7)) was associated independently with both neutralizing antibody and CMI responses. Protection was associated with SHIV(DH12 (Clone 7)) serum neutralizing antibody titers of > or =1:80 or with cellular immune responses corresponding to >2,000 spot forming cells per 10(6) peripheral blood mononuclear cells. Immunization was also associated with a reduction in the magnitude and duration of virus load. Induction of cross-reactive, primary HIV-1-neutralizing antibodies is feasible and, when potent, may result in complete protection against infection with a heterologous challenge virus strain.     

Polyvalent DNA prime and envelope protein boost HIV-1 vaccine elicits humoral and cellular responses and controls plasma viremia in rhesus macaques following rectal challenge with an R5 SHIV isolate

Immunization of macaques with multivalent DNA encoding gp120 genes from HIV-1 subtypes A, B, C and E and a gag gene followed by boosting with homologous gp120 proteins elicited strong anti-gp120 antibodies capable of neutralizing homologous and to a lesser degree heterologous HIV-1 isolates. Both Env- and Gag-specific cell mediated immune (CMI) responses were detected in the immunized animals. Following rectal challenge with an SHIV isolate encoding HIV-1(Ba-L)env, plasma viremia in the infected immunized animals was significantly lower than that observed in the na?ve animals. Further, one of six immunized animals was completely protected whereas all six na?ve animals were infected. These results demonstrate that a vaccine based on priming with a polyvalent DNA vaccine from multiple HIV-1 subtypes followed by boosting with homologous Env proteins elicits anti-HIV-1 immune responses capable of controlling rectal transmission of SHIV(Ba-L).     

Impact of the HIV-1 env genetic context outside HR1-HR2 on resistance to the fusion inhibitor enfuvirtide and viral infectivity in clinical isolates

Resistance mutations to the HIV-1 fusion inhibitor enfuvirtide emerge mainly within the drug's target region, HR1, and compensatory mutations have been described within HR2. The surrounding envelope (env) genetic context might also contribute to resistance, although to what extent and through which determinants remains elusive. To quantify the direct role of the env context in resistance to enfuvirtide and in viral infectivity, we compared enfuvirtide susceptibility and infectivity of recombinant viral pairs harboring the HR1-HR2 region or the full Env ectodomain of longitudinal env clones from 5 heavily treated patients failing enfuvirtide therapy. Prior to enfuvirtide treatment onset, no env carried known resistance mutations and full Env viruses were on average less susceptible than HR1-HR2 recombinants. All escape clones carried at least one of G36D, V38A, N42D and/or N43D/S in HR1, and accordingly, resistance increased 11- to 2800-fold relative to baseline. Resistance of full Env recombinant viruses was similar to resistance of their HR1-HR2 counterpart, indicating that HR1 and HR2 are the main contributors to resistance. Strictly X4 viruses were more resistant than strictly R5 viruses, while dual-tropic Envs featured similar resistance levels irrespective of the coreceptor expressed by the cell line used. Full Env recombinants from all patients gained infectivity under prolonged drug pressure; for HR1-HR2 viruses, infectivity remained steady for 3/5 patients, while for 2/5 patients, gains in infectivity paralleled those of the corresponding full Env recombinants, indicating that the env genetic context accounts mainly for infectivity adjustments. Phylogenetic analyses revealed that quasispecies selection is a step-wise process where selection of enfuvirtide resistance is a dominant factor early during therapy, while increased infectivity is the prominent driver under prolonged therapy.     

The Thai phase III trial (RV144) vaccine regimen induces T cell responses that preferentially target epitopes within the V2 region of HIV-1 envelope

The Thai HIV phase III prime/boost vaccine trial (RV144) using ALVAC-HIV (vCP1521) and AIDSVAX B/E was, to our knowledge, the first to demonstrate acquisition efficacy. Vaccine-induced, cell-mediated immune responses were assessed. T cell epitope mapping studies using IFN-γ ELISPOT was performed on PBMCs from HIV-1-uninfected vaccine (n = 61) and placebo (n = 10) recipients using HIV-1 Env peptides. Positive responses were measured in 25 (41%) vaccinees and were predominantly CD4(+) T cell-mediated. Responses were targeted within the HIV Env region, with 15 of 25 (60%) of vaccinees recognizing peptides derived from the V2 region of HIV-1 Env, which includes the α(4)β(7) integrin binding site. Intracellular cytokine staining confirmed that Env responses predominated (19 of 30; 63% of vaccine recipients) and were mediated by polyfunctional effector memory CD4(+) T cells, with the majority of responders producing both IL-2 and IFN-γ (12 of 19; 63%). HIV Env Ab titers were higher in subjects with IL-2 compared with those without IL-2-secreting HIV Env-specific effector memory T cells. Proliferation assays revealed that HIV Ag-specific T cells were CD4(+), with the majority (80%) expressing CD107a. HIV-specific T cell lines obtained from vaccine recipients confirmed V2 specificity, polyfunctionality, and functional cytolytic capacity. Although the RV144 T cell responses were modest in frequency compared with humoral immune responses, the CD4(+) T cell response was directed to HIV-1 Env and more particularly the V2 region.     

Differential regulation of antigen-specific CD8+ T cell responses by IL-12p40 in a dose-dependent manner

IL-12p40 is a natural antagonist which inhibits IL-12- and IL-23-mediated biological activity by blocking the binding of IL-12/23 to their receptors. Recently, IL-12p40 was also shown to have immune-enhancing activity through the activation of macrophages or dendritic cells. In this study, we investigated the effects of IL-12p40 as a genetic adjuvant on immune modulation using recombinant adenoviruses expressing IL-12p40 (rAd/IL-12p40) and OVA (rAd/OVA). Coimmunization of rAd/IL-12p40 at a low dose (1 x 10(4) PFU) with rAd/OVA resulted in OVA-specific immune enhancement, while a high dose of rAd/IL-12p40 (1 x 10(8) PFU) caused significant suppression of CD8(+) T cell responses. In addition, the enhancement and suppression of OVA-specific CD8(+) T cell responses correlated with antitumor activity against E.G7-OVA tumor challenge, which subsequently affected the survival rate. Moreover, the differential CD8(+) T cell response by IL-12p40 was still observed in IL-12Rbeta2 knockout (IL-12Rbeta2KO), but not in IL-12Rbeta1 knockout (IL-12Rbeta1KO) mice, indicating that IL-12p40 is a cytokine which can modulate Ag-specific T cell responses depending on IL-12Rbeta1. Our findings provide a novel insight on the physiological role of IL-12p40, which can be informative in the design of vaccine strategies and therapeutic regimens.     

Increased level and longevity of protective immune responses induced by DNA vaccine expressing the HIV-1 Env glycoprotein when combined with IL-21 and IL-15 gene delivery

We investigated the ability of a plasmid-derived IL-21 delivered alone or in combination with the IL-15 gene to regulate immune responses to the HIV-1 envelope (Env) glycoprotein induced by DNA vaccination. Mice were injected with the gp140DeltaCFI(HXB2/89.6) vector expressing a modified Env glycoprotein with C-terminal mutations intended to mimic a fusion intermediate, in which the most divergent region encoding the variable V1, V2, and V3 domains of CXCR4-tropic HxB2 virus was replaced with the dual-tropic 89.6 viral strain. Using a recombinant vaccinia virus expressing 89.6 Env glycoprotein (vBD3) in a mouse challenge model, we observed that IL-21 plasmid produced sustained resistance to viral transmission when injected 5 days after DNA vaccination. Moreover, IL-21 in a synergistic manner with IL-15 expression vector augmented the vaccine-induced recall responses to the vBD3 challenge compared with those elicited by immunization in the presence of either cytokine alone. The synergistic combination of IL-21 and IL-15 plasmids promoted expansion of CD8+CD127+ memory T cell pools specific for a subdominant HLA-A2-restricted Env(121-129) epitope (KLTPLCVTL). Our results also show that coimmunization with IL-21 and IL-15 plasmid combination resulted in enhanced CD8+ T cell function that was partially independent of CD4+ T cell help in mediating protection against vBD3 challenge. Furthermore, the use of IL-21 and IL-15 genes was able to increase Ab-dependent cellular cytotoxicity and complement-dependent lysis of Env-expressing target cells through augmentation of Env-specific IgG Ab levels. These data indicate that the plasmid-delivered IL-21 and IL-15 can increase the magnitude of the response to DNA vaccines.     

The genetic bottleneck in vertical transmission of subtype C HIV-1 is not driven by selection of especially neutralization-resistant virus from the maternal viral population

Subtype C human immunodeficiency virus type 1 (HIV-1C) continues to cause the majority of new cases of mother-to-child transmission (MTCT), and yet there are limited data on HIV-1C transmission. We amplified env from plasma RNA for 19 HIV-1C MTCT pairs, 10 transmitting in utero (IU) and 9 transmitting intrapartum (IP). There was a strong genetic bottleneck between all mother-infant pairs, with a majority of transmission events involving the transmission of a single virus. env genes of viruses transmitted to infants IP, but not IU, encoded Env proteins that were shorter and had fewer putative N-linked glycosylation sites in the V1-V5 region than matched maternal sequences. Viruses pseudotyped with env clones representative of each maternal and infant population were tested for neutralization sensitivity. The 50% inhibitory concentration of autologous serum was similar against both transmitted (infant) and nontransmitted (maternal) viruses in a paired analysis. Mother and infant Env proteins were also similar in sensitivity to soluble CD4, to a panel of monoclonal antibodies, and to heterologous HIV-1C sera. In addition, there was no difference in the breadth or potency of neutralizing antibodies between sera from 50 nontransmitting and 23 IU and 23 IP transmitting HIV-1C-infected women against four Env proteins from heterologous viruses. Thus, while a strong genetic bottleneck was detected during MCTC, with viruses of shorter and fewer glycosylation sites in env present in IP transmission, our data do not support this bottleneck being driven by selective resistance to antibodies.     

Stoichiometry of Monoclonal Antibody Neutralization of T-Cell Line-Adapted Human Immunodeficiency Virus Type 1

In order to study the stoichiometry of monoclonal antibody (MAb) neutralization of T-cell line-adapted human immunodeficiency virus type 1 (HIV-1) in antibody excess and under equilibrium conditions, we exploited the ability of HIV-1 to generate mixed oligomers when different env genes are coexpressed. By the coexpression of Env glycoproteins that either can or cannot bind a neutralizing MAb in an env transcomplementation assay, virions were generated in which the proportion of MAb binding sites could be regulated. As the proportion of MAb binding sites in Env chimeric virus increased, MAb neutralization gradually increased. Virus neutralization by virion aggregation was minimal, as MAb binding to HIV-1 Env did not interfere with an AMLV Env-mediated infection by HIV-1(AMLV/HIV-1) pseudotypes of CD4(-) HEK293 cells. MAb neutralization of chimeric virions could be described as a third-order function of the proportion of Env antigen refractory to MAb binding. This scenario is consistent with the Env oligomer constituting the minimal functional unit and neutralization occurring incrementally as each Env oligomer binds MAb. Alternatively, the data could be fit to a sigmoid function. Thus, these data could not exclude the existence of a threshold for neutralization. However, results from MAb neutralization of chimeric virus containing wild-type Env and Env defective in CD4 binding was readily explained by a model of incremental MAb neutralization. In summary, the data indicate that MAb neutralization of T-cell line-adapted HIV-1 is incremental rather than all or none and that each MAb binding an Env oligomer reduces the likelihood of infection.     

Roles of vaccinia virus genes E3L and K3L and host genes PKR and RNase L during intratracheal infection of C57BL/6 mice

The importance of the 2'-5' oligoadenylate synthetase (OAS)/RNase L and double-stranded RNA (dsRNA)-dependent protein kinase (PKR) pathways in host interferon induction resulting from virus infection in response to dsRNA has been well documented. In poxvirus infections, the interactions between the vaccinia virus (VV) genes E3L and K3L, which target RNase L and PKR, respectively, serve to prevent the induction of the dsRNA-dependent induced interferon response in cell culture. To determine the importance of these host genes in controlling VV infections, mouse single-gene knockouts of RNase L and PKR and double-knockout mice were studied following intratracheal infection with VV, VVΔK3L, or VVΔE3L. VV caused lethal disease in all mouse strains. The single-knockout animals were more susceptible than wild-type animals, while the RNase L(-/-) PKR(-/-) mice were the most susceptible. VVΔE3L infections of wild-type mice were asymptomatic, demonstrating that E3L plays a critical role in controlling the host immune response. RNase L(-/-) mice showed no disease, whereas 20% of the PKR(-/-) mice succumbed at a dose of 10(8) PFU. Lethal disease was routinely observed in RNase L(-/-) PKR(-/-) mice inoculated with 10(8) PFU of VVΔE3L, with a distinct pathology. VVΔK3L infections exhibited no differences in virulence among any of the mouse constructs, suggesting that PKR is not the exclusive target of K3L. Surprisingly, VVΔK3L did not disseminate to other tissues from the lung. Hence, the cause of death in this model is respiratory disease. These results also suggest that an unanticipated role of the K3L gene is to facilitate virus dissemination.     

Deciphering human immunodeficiency virus type 1 transmission and early envelope diversification by single-genome amplification and sequencing

Accurate identification of the transmitted virus and sequences evolving from it could be instrumental in elucidating the transmission of human immunodeficiency virus type 1 (HIV-1) and in developing vaccines, drugs, or microbicides to prevent infection. Here we describe an experimental approach to analyze HIV-1 env genes as intact genetic units amplified from plasma virion RNA by single-genome amplification (SGA), followed by direct sequencing of uncloned DNA amplicons. We show that this strategy precludes in vitro artifacts caused by Taq-induced nucleotide substitutions and template switching, provides an accurate representation of the env quasispecies in vivo, and has an overall error rate (including nucleotide misincorporation, insertion, and deletion) of less than 8 x 10(-5). Applying this method to the analysis of virus in plasma from 12 Zambian subjects from whom samples were obtained within 3 months of seroconversion, we show that transmitted or early founder viruses can be identified and that molecular pathways and rates of early env diversification can be defined. Specifically, we show that 8 of the 12 subjects were each infected by a single virus, while 4 others acquired more than one virus; that the rate of virus evolution in one subject during an 80-day period spanning seroconversion was 1.7 x 10(-5) substitutions per site per day; and that evidence of strong immunologic selection can be seen in Env and overlapping Rev sequences based on nonrandom accumulation of nonsynonymous mutations. We also compared the results of the SGA approach with those of more-conventional bulk PCR amplification methods performed on the same patient samples and found that the latter is associated with excessive rates of Taq-induced recombination, nucleotide misincorporation, template resampling, and cloning bias. These findings indicate that HIV-1 env genes, other viral genes, and even full-length viral genomes responsible for productive clinical infection can be identified by SGA analysis of plasma virus sampled at intervals typical in large-scale vaccine trials and that pathways of viral diversification and immune escape can be determined accurately.     

Suppression of hyperglycemia in NOD mice after inoculation with recombinant vaccinia viruses

In autoimmune (type 1) diabetes, autoreactive lymphocytes destroy pancreatic β-cells responsible for insulin synthesis. To assess the feasibility of gene therapy for type 1 diabetes, recombinant vaccinia virus (rVV) vectors were constructed expressing pancreatic islet autoantigens proinsulin (INS) and a 55-kDa immunogenic peptide from glutamic acid decarboxylase (GAD), and the immunomodulatory cytokine interleukin (IL)-10. To augment the beneficial effects of recombinant virus therapy, the INS and GAD genes were fused to the C terminus of the cholera toxin B subunit (CTB). Five-week-old non-obese diabetic (NOD) mice were injected once with rVV. Humoral antibody immune responses and hyperglycemia in the infected mice were analyzed. Only 20% of the mice inoculated with rVV expressing the CTB::INS fusion protein developed hyperglycemia, in comparison to 70% of the mice in the uninoculated animal group. Islets from pancreatic tissues isolated from euglycemic mice from this animal group showed no sign of inflammatory lymphocyte invasion. Inoculation with rVV producing CTB::GAD or IL-10 was somewhat less effective in reducing diabetes. Humoral antibody isotypes of hyperglycemic and euglycemic mice from all treated groups possessed similar IgG1/IgG2c antibody titer ratios from 19 to 32 wk after virus inoculation. In comparison with uninoculated mice, 11-wk-old NOD mice injected with virus expressing CTB::INS were delayed in diabetes onset by more than 4 wk. The experimental results demonstrate the feasibility of using rVV expressing CTB::INS fusion protein to generate significant protection and therapy against type 1 diabetes onset and progression.     

Characterization of recombinant nonecotropic murine leukemia viruses from the wild mouse species Mus spretus

The wild mouse species most closely related to the common laboratory strains contain proviral env genes of the xenotropic/polytropic subgroup of mouse leukemia viruses (MLVs). To determine if the polytropic proviruses of Mus spretus contain functional genes, we inoculated neonates with Moloney MLV (MoMLV) or amphotropic MLV (A-MLV) and screened for viral recombinants with altered host ranges. Thymus and spleen cells from MoMLV-inoculated mice were plated on Mus dunni cells and mink cells, since these cells do not support the replication of MoMLV, and cells from A-MLV-inoculated mice were plated on ferret cells. All MoMLV-inoculated mice produced ecotropic viruses that resembled their MoMLV progenitor, although some isolates, unlike MoMLV, grew to high titers in M. dunni cells. All of the MoMLV-inoculated mice also produced nonecotropic virus that was infectious for mink cells. Sequencing of three MoMLV- and two A-MLV-derived nonecotropic recombinants confirmed that these viruses contained substantial substitutions that included the regions of env encoding the surface (SU) protein and the 5' end of the transmembrane (TM) protein. The 5' recombination breakpoint for one of the A-MLV recombinants was identified in RNase H. The M. spretus-derived env substitutions were nearly identical to the corresponding regions in prototypical laboratory mouse polytropic proviruses, but the wild mouse infectious viruses had a more restricted host range. The M. spretus proviruses contributing to these recombinants were also sequenced. The seven sequenced proviruses were 99% identical to one another and to the recombinants; only two of the seven had obvious fatal defects. We conclude that the M. spretus proviruses are likely to be recent germ line acquisitions and that they contain functional genes that can contribute to the production of replication-competent virus.     

Robust recall and long-term memory T-cell responses induced by prime-boost regimens with heterologous live viral vectors expressing human immunodeficiency virus type 1 Gag and Env proteins

We investigated long-term memory and recall cellular immune responses to human immunodeficiency virus type 1 (HIV-1) Env and Gag proteins elicited by recombinant vesicular stomatitis viruses (VSVs) expressing Env and Gag. More than 7 months after a single vaccination with VSV-Env, approximately 6% of CD8(+) splenocytes stained with major histocompatibility complex class I tetramers containing the Env p18-I10 immunodominant peptide and showed a memory phenotype (CD44(Hi)). The level of tetramer-positive cells in memory was about 14% of the peak primary response. Recall responses elicited in these mice 5 days after boosting with a heterologous recombinant vaccinia virus expressing HIV-1 Env showed that 40 to 45% of CD8(+) splenocytes were tetramer positive and activated (CD62L(Lo)), and these cells produced gamma interferon after stimulation with Env peptide, indicating that they were functional. Five months after the boost, the long-term memory cell population (tetramer positive, CD44(Hi)) constituted 30% of the CD8(+) splenocytes. Recall responses to HIV-1 Gag were examined in mice primed with VSV recombinants expressing HIV-1 Gag protein and boosted with a vaccinia virus recombinant expressing Gag. Using this protocol, we found that approximately 40% of CD8(+) splenocytes were activated (CD62L(Lo)) and specific for a Gag immunodominant peptide (tetramer positive). The high-level Gag recall response elicited by the vaccinia virus-Gag was greater than that obtained by boosting with a VSV-Gag vector with a different VSV glycoprotein. The corresponding levels of CD44(Hi) memory cells were also higher long after boosting with vaccinia virus-Gag than after boosting with a glycoprotein exchange VSV-Gag. Our results show that VSV vectors elicit high-level memory CTL responses and that these can be amplified as much as six- to sevenfold using a heterologous boosting vector.