CD4-Pseudomonas exotoxin conjugates delay but do not fully inhibit human immunodeficiency virus replication in lymphocytes in vitro.

The CD4 molecule is a high affinity receptor for the human immunodeficiency virus (HIV) envelope glycoprotein (gp160 or gp120). This glycoprotein is expressed on the surface membrane of cells infected with HIV. It has, therefore, been suggested that a soluble form of CD4 might be used as a targeting agent to deliver toxins selectively to cells infected with HIV. We demonstrate that CD4-Pseudomonas exotoxin A (PE) conjugates inhibit the proliferation of gp160-transfected Chinese hamster ovary cells and block HIV replication in virus-infected H9 cells. However, this inhibition of HIV replication appears to be incomplete since virus replication occurs following removal of the toxin conjugates from these cultures. Moreover, CD4-PE conjugates delay but do not inhibit HIV replication in human peripheral blood lymphocytes. These studies suggest that such conjugates should be assessed only as potential adjunctive therapies in the acquired immunodeficiency syndrome.     

Lymphomatoid papulosis and human herpesviruses--A PCR-based evaluation for the presence of human herpesvirus 6, 7 and 8 related herpesviruses

BACKGROUND: Lymphomatoid papulosis (LyP) is a chronic, recurrent lymphoproliferative disorder of the skin that belongs to the group of primary cutaneous CD30-positive T-cell lymphomas. Ultrastructural and clinical features of LyP suggest that it has a viral etiology. Human herpesviruses have been proposed as causative cofactors for LyP because of their oncogenic potential and their association with other lymphomas. METHODS: LyP skin lesions and a LyP-derived cell line were examined for the presence of the recently discovered oncogenic human herpesvirus 8 (HHV-8) and the two T-lymphotropic human herpesviruses 6 and 7 (HHV-6 and HHV-7) by nested polymerase chain reaction (PCR) using virus-specific oligonucleotide primers. Furthermore, a recently described method involving degenerate PCR primers was applied to detect highly conserved DNA sequences shared by a variety of herpesviruses, especially oncogenic gamma-herpesviruses, in an attempt to identify a yet undiscovered herpesvirus associated with LyP. RESULTS: HHV-6 and 8 could not be found in 26 archival and 11 snap-frozen LyP lesions and a LyP tumor cell line. HHV-7 DNA sequences were detected in 14% (5 of 37) of LyP samples. HHV-6 was found in 23% (3 of 13) and HHV-7 in 8% (1 of 13) of normal skin samples from healthy individuals, respectively. Using degenerate PCR primers to amplify the highly conserved polymerase region of herpesviruses, no DNA sequences related to human herpesviruses could be detected. CONCLUSIONS: LyP is not associated with HHV-6, HHV-7 and HHV-8. In addition, the studies using degenerate PCR primers do not indicate the presence of a previously undescribed human herpesvirus in LyP.     

Anti-gamma interferon antibodies enhance the immunogenicity of recombinant adenovirus vectors

Vaccination for eliciting antigen-specific memory CD8(+) T cells may be facilitated by manipulating the pleiotropic effects of gamma interferon (IFN-γ). We assessed strategies for modulating the contribution of IFN-γ during the development of antigen-specific cytotoxic T lymphocyte (CTL) populations. We first showed that recombinant IFN-γ suppressed antigen expression in vitro from a recombinant adenovirus (rAd) vector in a dose-dependent manner and that addition of an anti-IFN-γ antibody (Ab) eliminated this suppression. Consistent with these in vitro findings, we found that HIV-1 envelope (Env)-specific CTL responses were higher in IFN-γ-knockout (GKO) mice than in wild-type mice following immunization with rAd. Since these observations suggested that IFN-γ might suppress rAd-induced CTL development, we assessed the ability of anti-IFN-γ Ab administration to augment rAd-elicited CTL in vivo. In fact, blockage of IFN-γ activity by monoclonal Ab administration was associated with elevated levels of interleukin 7 receptor alpha chain-positive (IL-7Rα(+)) Env-specific CTL populations postboost. These observations illustrate the utility of an anti-IFN-γ Ab for potentiating rAd immunizations to effect quantitative and qualitative changes in the effector and memory CTL populations.     

Intranasal vaccination with the recombinant Listeria monocytogenes ΔactA prfA* mutant elicits robust systemic and pulmonary cellular responses and secretory mucosal IgA

We previously showed that recombinant (r) Listeria monocytogenes carrying ΔactA and a selected prfA* mutation (r-Listeria ΔactA prfA*) secreted >100-fold more immunogen in broth culture than wild-type r-Listeria or r-Listeria ΔactA and elicited much greater cellular and humoral immune responses than r-Listeria ΔactA after intravenous vaccination of mice. Here, we conducted comparative studies evaluating vaccine-elicited immune responses in systemic and mucosal sites after intranasal, intravenous, intraperitoneal, or subcutaneous immunization of mice with r-Listeria ΔactA prfA* vaccine candidates. Intranasal vaccination of mice with r-Listeria ΔactA prfA* vaccine candidates elicited a robust gamma interferon-positive (IFN-γ(+)) cellular response in systemic sites, although intravenous or intraperitoneal immunization was more efficient. Surprisingly, intranasal vaccination elicited an appreciable pulmonary IFN-γ(+) cellular response that was nonstatistically higher than the magnitude induced by the intravenous route but was significantly greater than that elicited by subcutaneous immunization. Furthermore, although intranasal r-Listeria ΔactA prfA* delivery induced poor systemic IgG responses, intranasal vaccination elicited appreciable secretory immunogen-specific IgA titers that were similar to or higher in mucosal fluid than those induced by subcutaneous and intravenous immunizations. Thus, intranasal vaccination with r-Listeria ΔactA prfA* appears to be a useful approach for eliciting robust systemic and pulmonary cellular responses and measurable secretory mucosal IgA titers.     

Immune and Genetic Correlates of Vaccine Protection Against Mucosal Infection by SIV in Monkeys

The RV144 vaccine trial in Thailand demonstrated that an HIV vaccine could prevent infection in humans and highlights the importance of understanding protective immunity against HIV. We used a nonhuman primate model to define immune and genetic mechanisms of protection against mucosal infection by the simian immunodeficiency virus (SIV). A plasmid DNA prime/recombinant adenovirus serotype 5 (rAd5) boost vaccine regimen was evaluated for its ability to protect monkeys from infection by SIVmac251 or SIVsmE660 isolates after repeat intrarectal challenges. Although this prime-boost vaccine regimen failed to protect against SIVmac251 infection, 50% of vaccinated monkeys were protected from infection with SIVsmE660. Among SIVsmE660-infected animals, there was about a one-log reduction in peak plasma virus RNA in monkeys expressing the major histocompatibility complex class I allele Mamu-A*01, implicating cytotoxic T lymphocytes in the control of SIV replication once infection is established. Among Mamu-A*01-negative monkeys challenged with SIVsmE660, no CD8(+) T cell response or innate immune response was associated with protection against virus acquisition. However, low levels of neutralizing antibodies and an envelope-specific CD4(+) T cell response were associated with vaccine protection in these monkeys. Moreover, monkeys that expressed two TRIM5 alleles that restrict SIV replication were more likely to be protected from infection than monkeys that expressed at least one permissive TRIM5 allele. This study begins to elucidate the mechanisms of vaccine protection against immunodeficiency viruses and highlights the need to analyze these immune and genetic correlates of protection in future trials of HIV vaccine strategies.     

CD8+ cytotoxic T lymphocyte responses to lentiviruses and herpesviruses

Immune containment of persistent viral infections has long been a focus of interest for investigators. However, the technologies needed to evaluate the role of CD8+ cytotoxic T lymphocytes (CTLs) in this process have only recently become available. Recent studies performed using tetramer, ELISPOT and cytokine-production assays have evaluated the role of CD8+ CTLs in controlling lentivirus and herpesvirus infections in humans and nonhuman primates. These studies demonstrate dramatic expansions of virus-specific CTLs in primary infection and the maintenance of unexpectedly high levels of virus-specific CTLs in chronic infection. These findings underscore the importance of CD8+ CTLs in the immune control of persistent viral infections.     

Immunization with HIV-1 SF162-derived Envelope gp140 proteins does not protect macaques from heterologous simian-human immunodeficiency virus SHIV89.6P infection

Immunization by the SF162gp140 or the DeltaV2gp140 HIV-1 envelope proteins results in the generation of strong homologous neutralizing antibodies (NAbs) that offer similar degree of protection from disease-development to macaques challenged with homologous virus. These two immunogens elicit weak cross-reactive NAbs and their effectiveness against heterologous challenge is currently unknown. To examine this issue, we immunized macaques with SIVGag p55 and either the SF162gp140 or the DeltaV2gp140 and challenged them intravenously with SHIV-89.6P. All animals became infected but previous immunization with SF162gp140 accelerated the development of anti-SHIV89.6P neutralizing antibody responses following infection. DeltaV2gp140 is derived from SF162gp140 following the deletion of 30 amino acids and one N-linked glycosylation site from the V2 loop. Our results suggest that even small differences in HIV Envelope immunogen structure can affect the neutralizing antibody responses generated following infection.     

Inhibition of allergic encephalomyelitis in marmosets by vaccination with recombinant vaccinia virus encoding for myelin basic protein

A primary demyelinating form of experimental allergic encephalomyelitis (EAE) resembling human multiple sclerosis (MS) occurs in Callithrix jacchus marmosets following immunization with human white matter. Participation of a T-cell immune response against myelin basic protein (MBP) in this disease model is supported by observations of increased reactivity against MBP in PBMC and of adoptive transfer of an inflammatory form of EAE by MBP-reactive T-cells. To evaluate the effects of ectopic presentation of MBP on marmoset EAE, animals were vaccinated prior to induction of EAE by subcutaneous injection of attenuated strains of vaccinia virus genetically engineered to contain either the entire coding sequence for human MBP (vT15) or the equine herpes virus glycoprotein gH gene (vAbT249). Vaccination with vT15 was followed by transient cytoplasmic and surface membrane expression of MBP in circulating PBMC (15–45 days). The onset of clinical EAE after immunization (pi) was markedly delayed in vT15-vaccinated animals (37–97 days pi, n=4) compared to vAbT249-vaccinated controls (14–18 days pi, n=3). Proliferative responses against MBP but not against vaccinia antigens or phytohemagglutinin were suppressed in protected animals. Thus, development of attenuated live viruses carrying genes for myelin antigens could be useful for induction of immunologic tolerance and for modulation of autoimmune demyelination.     

A centralized gene-based HIV-1 vaccine elicits broad cross-clade cellular immune responses in rhesus monkeys

One of the major challenges that must be met in developing an HIV-1 vaccine is devising a strategy to generate cellular immunity with sufficient breadth to deal with the extraordinary genetic diversity of the virus. Amino acids in the envelopes of viruses from the same clade can differ by >15%, and those from different clades can differ by >30%. It has been proposed that creating immunogens using centralized HIV-1 gene sequences might provide a practical solution to this problem. Such centralized genes can be generated by employing a number of different strategies: consensus, ancestral, or center of tree sequences. These computer-generated sequences are a shorter genetic distance from any two contemporary virus sequences than those contemporary sequences are from each other. The present study was initiated to evaluate the breadth of cellular immunity generated through immunization of rhesus monkeys with vaccine constructs expressing either an HIV-1 global consensus envelope sequence (CON-S) or a single patient isolate clade B envelope sequence (clade B). We show that vaccine immunogens expressing the single centralized gene CON-S generated cellular immune responses with significantly increased breadth compared with immunogens expressing a wild-type virus gene. In fact, CON-S immunogens elicited cellular immune responses to 3- to 4-fold more discrete epitopes of the envelope proteins from clades A, C, and G than did clade B immunogens. These findings suggest that immunization with centralized genes is a promising vaccine strategy for developing a global vaccine for HIV-1 as well as vaccines for other genetically diverse viruses.