BAC-VAC, a novel generation of (DNA) vaccines: A bacterial artificial chromosome (BAC) containing a replication-competent, packaging-defective virus genome induces protective immunity against herpes simplex virus 1

This study aimed to exploit bacterial artificial chromosomes (BAC) as large antigen-capacity DNA vaccines (BAC-VAC) against complex pathogens, such as herpes simplex virus 1 (HSV-1). The 152-kbp HSV-1 genome recently has been cloned as an F-plasmid-based BAC in Escherichia coli (fHSV), which can efficiently produce infectious virus progeny upon transfection into mammalian cells. A safe modification of fHSV, fHSVΔpac, does not give rise to progeny virus because the signals necessary to package DNA into virions have been excluded. However, in mammalian cells fHSVΔpac DNA can still replicate, express the HSV-1 genes, cause cytotoxic effects, and produce virus-like particles. Because these functions mimic the lytic cycle of the HSV-1 infection, fHSVΔpac was expected to stimulate the immune system as efficiently as a modified live virus vaccine. To test this hypothesis, mice were immunized with fHSVΔpac DNA applied intradermally by gold-particle bombardment, and the immune responses were compared with those induced by infection with disabled infectious single cycle HSV-1. Immunization with either fHSVΔpac or disabled infectious single cycle HSV-1 induced the priming of HSV-1-specific cytotoxic T cells and the production of virus-specific antibodies and conferred protection against intracerebral injection of wild-type HSV-1 at a dose of 200 LD50. Protection probably was cell-mediated, as transfer of serum from immunized mice did not protect naive animals. We conclude that BAC-VACs per se, or in combination with genetic elements that support replicative amplification of the DNA in the cell nucleus, represent a useful new generation of DNA-based vaccination strategies for many viral and nonviral antigens.

Inhibition of luciferase expression in transgenic Aedes aegypti mosquitoes by Sindbis virus expression of antisense luciferase RNA

A rapid and reproducible method of inhibiting the expression of specific genes in mosquitoes should further our understanding of gene function and may lead to the identification of mosquito genes that determine vector competence or are involved in pathogen transmission. We hypothesized that the virus expression system based on the mosquito-borne Alphavirus, Sindbis (Togaviridae), may efficiently transcribe effector RNAs that inhibit expression of a targeted mosquito gene. To test this hypothesis, germ-line-transformed Aedes aegypti that express luciferase (LUC) from the mosquito Apyrase promoter were intrathoracically inoculated with a double subgenomic Sindbis (dsSIN) virus TE/3′2J/anti-luc (Anti-luc) that transcribes RNA complementary to the 5′ end of the LUC mRNA. LUC activity was monitored in mosquitoes infected with either Anti-luc or control dsSIN viruses expressing unrelated antisense RNAs. Mosquitoes infected with Anti-luc virus exhibited 90% reduction in LUC compared with uninfected and control dsSIN-infected mosquitoes at 5 and 9 days postinoculation. We demonstrate that a gene expressed from the mosquito genome can be inhibited by using an antisense strategy. The dsSIN antisense RNA expression system is an important tool for studying gene function in vivo.

Ecological factors rather than temporal factors dominate the evolution of vesicular stomatitis virus

Vesicular stomatitis New Jersey virus (VSV-NJ) is a rhabdovirus that causes economically important disease in cattle and other domestic animals in endemic areas from southeastern United States to northern South America. Its negatively stranded RNA genome is capable of undergoing rapid evolution, which allows phylogenetic analysis and molecular epidemiology studies to be performed. Previous epidemiological studies in Costa Rica showed the existence of at least two distinct ecological zones of high VSV-NJ activity, one located in the highlands (premontane tropical moist forest) and the other in the lowlands (tropical dry forest). We wanted to test the hypothesis that the viruses circulating in these ecological zones were genetically distinct. For this purpose, we sequenced the hypervariable region of the phosphoprotein gene for 50 VSV-NJ isolates from these areas. Phylogenetic analysis showed that viruses from each ecological zone had distinct genotypes. These genotypes were maintained in each area for periods of up to 8 years. This evolutionary pattern of VSV-NJ suggests an adaptation to ecological factors that could exert selective pressure on the virus. As previous data indicated an absence of virus adaptation to factors related to the bovine host (including immunological pressure), it appears that VSV genetic divergence represents positive selection to adapt to specific vectors and/or reservoirs at each ecological zone.

Prevention of hepatitis C virus infection in chimpanzees by hyperimmune serum against the hypervariable region 1 of the envelope 2 protein

The identification of the neutralization domains of hepatitis C virus (HCV) is essential for the development of an effective vaccine. Here, we show that the hypervariable region 1 (HVR1) of the envelope 2 (E2) protein is a critical neutralization domain of HCV. Neutralization of HCV in vitro was attempted with a rabbit hyperimmune serum raised against a homologous synthetic peptide derived from the HVR1 of the E2 protein, and the residual infectivity was evaluated by inoculation of HCV-seronegative chimpanzees. The source of HCV was plasma obtained from a patient (H) during the acute phase of posttransfusion non-A, non-B hepatitis, which had been titered for infectivity in chimpanzees. The anti-HVR1 antiserum induced protection against homologous HCV infection in chimpanzees, but not against the emergence of neutralization escape mutants that were found to be already present in the complex viral quasispecies of the inoculum. The finding that HVR1 can elicit protective immunity opens new perspectives for the development of effective preventive strategies. However, the identification of the most variable region of HCV as a critical neutralization domain poses a major challenge for the development of a broadly reactive vaccine against HCV.

Prophylactic DNA vaccine for hepatitis C virus (HCV) infection: HCV-specific cytotoxic T lymphocyte induction and protection from HCV-recombinant vaccinia infection in an HLA-A2.1 transgenic mouse model

DNA vaccines express antigens intracellularly and effectively induce cellular immune responses. Because only chimpanzees can be used to model human hepatitis C virus (HCV) infections, we developed a small-animal model using HLA-A2.1-transgenic mice to test induction of HLA-A2.1-restricted cytotoxic T lymphocytes (CTLs) and protection against recombinant vaccinia expressing HCV-core. A plasmid encoding the HCV-core antigen induced CD8+ CTLs specific for three conserved endogenously expressed core peptides presented by human HLA-A2.1. When challenged, DNA-immunized mice showed a substantial (5–12 log10) reduction in vaccinia virus titer compared with mock-immunized controls. This protection, lasting at least 14 mo, was shown to be mediated by CD8+ cells. Thus, a DNA vaccine expressing HCV-core is a potential candidate for a prophylactic vaccine for HLA-A2.1+ humans.

Tissue-specific expression of herpes simplex virus thymidine kinase gene delivered by adeno-associated virus inhibits the growth of human hepatocellular carcinoma in athymic mice

About 70% of hepatocellular carcinomas are known to express α-fetoprotein, which is normally expressed in fetal but not in adult livers. To induce herpes simplex virus-thymidine kinase expression in these cancer cells, we constructed an adeno-associated viral vector containing the HSV-TK gene under the control of the α-fetoprotein enhancer and albumin promoter. We previously demonstrated in vitro that although this vector can transduce a variety of human cells, only transduced AFP and albumin-expressing hepatocellular carcinoma cell lines were sensitive to killing by ganciclovir (GCV). In the present study, we explored the effect of this vector on hepatocellular carcinoma cells in vivo. Subcutaneous tumors generated in nude mice by implanting hepatocellular carcinoma cells previously transduced with this vector shrank dramatically after treatment with GCV. Bystander effect was also observed on the tumors generated by mixing transduced and untransduced cells. To test whether the tumor cells can be transduced by the virus in vivo, we injected the recombinant adeno-associated virus into tumors generated by untransduced hepatocarcinoma cell line. Tumor growth were retarded after treatment with GCV. These experiments demonstrate the feasibility of in vivo transduction of tumor cell with rAAV.

Midbrain injection of recombinant adeno-associated virus encoding rat glial cell line-derived neurotrophic factor protects nigral neurons in a progressive 6-hydroxydopamine-induced degeneration model of Parkinson’s disease in rats

A recombinant adeno-associated virus (rAAV) vector capable of infecting cells and expressing rat glial cell line-derived neurotrophic factor (rGDNF), a putative central nervous system dopaminergic survival factor, under the control of a potent cytomegalovirus (CMV) immediate/early promoter (AAV-MD-rGDNF) was constructed. Two experiments were performed to evaluate the time course of expression of rAAV-mediated GDNF protein expression and to test the vector in an animal model of Parkinson’s disease. To evaluate the ability of rAAV-rGDNF to protect nigral dopaminergic neurons in the progressive Sauer and Oertel 6-hydroxydopamine (6-OHDA) lesion model, rats received perinigral injections of either rAAV-rGDNF virus or rAAV-lacZ control virus 3 weeks prior to a striatal 6-OHDA lesion and were sacrificed 4 weeks after 6-OHDA. Cell counts of back-labeled fluorogold-positive neurons in the substantia nigra revealed that rAAV-MD-rGDNF protected a significant number of cells when compared with cell counts of rAAV-CMV-lacZ-injected rats (94% vs. 51%, respectively). In close agreement, 85% of tyrosine hydroxylase-positive cells remained in the nigral rAAV-MD-rGDNF group vs. only 49% in the lacZ group. A separate group of rats were given identical perinigral virus injections and were sacrificed at 3 and 10 weeks after surgery. Nigral GDNF protein expression remained relatively stable over the 10 weeks investigated. These data indicate that the use of rAAV, a noncytopathic viral vector, can promote delivery of functional levels of GDNF in a degenerative model of Parkinson’s disease.

Early events in poliovirus infection: virus-receptor interactions.

The interaction of poliovirus with its cell receptor initiates conformational changes that lead to uncoating of the viral RNA. Three types of genetic analyses have been used to study the poliovirus-receptor interaction: (i) mutagenesis of the poliovirus receptor (PVR), (ii) selection of viral mutants resistant to neutralization with soluble PVR, and (iii) selection of viral variants adapted to use mutant PVRs. The results of these studies show that a small portion of the first immunoglobulin-like domain of PVR contacts viral residues within a deep depression on the surface of the capsid that encircles the fivefold axis of symmetry. Viral capsid residues that influence the interaction with PVR are also found in locations such as the capsid interior that cannot directly contact PVR. These mutations might influence the ability of the capsid to undergo receptor-mediated conformational transitions that are necessary for high-affinity interactions with PVR.

Whole body positron emission tomography imaging of simian immunodeficiency virus-infected rhesus macaques.

Pathogenesis of simian immunodeficiency virus (SIV) infection in rhesus macaques begins with acute viremia and then progresses to a distributed infection in the solid lymphoid tissues, which is followed by a process of cellular destruction leading to terminal disease and death. Blood and tissue specimens show the progress of infection at the cellular level but do not reveal the pattern of infection and host responses occurring throughout the body. The purpose of this investigation was to determine whether positron emission tomography (PET) imaging with intravenous 2-18F-2-deoxyglucose (FDG) could identify activated lymphoid tissues in a living animal and whether this pattern would reflect the extent of SIV infection. PET images from SIV-infected animals were distinguishable from uninfected controls and revealed a pattern consistent with widespread lymphoid tissue activation. Significant FDG accumulation in colon along with mesenteric and ileocaecal lymph nodes was found in SIV infection, especially during terminal disease stages. Areas of elevated FDG uptake in the PET images were correlated with productive SIV infection using in situ hybridization as a test for virus replication. PET-FDG images of SIV-infected animals correlated sites of virus replication with high FDG accumulation. These data show that the method can be used to evaluate the distribution and activity of infected tissues in a living animal without biopsy. Fewer tissues had high FDG uptake in terminal animals than midstage animals, and both were clearly distinguishable from uninfected animal scans.

Human immunodeficiency virus type-1 Tat is an integral component of the activated transcription-elongation complex.

The human immunodeficiency virus type 1 transactivator protein, Tat, stimulates transcriptional elongation from the viral long terminal repeat. To test whether Tat associates directly with activated transcription complexes, we have used the lac repressor protein (LacR) to "trap" elongating RNA polymerases. The arrested transcription complexes were purified by binding biotinylated templates to streptaviridin-coated magnetic beads. Transcription complexes were released from the magnetic beads following cleavage of the templates with restriction enzymes and were immunoblotted with antibodies to Tat, LacR and RNA polymerase II. The Tat protein copurified with RNA polymerase bound to wild-type templates but did not copurify with transcription complexes prepared by using templates carrying mutations in the transactivation response element (TAR) RNA. We conclude that Tat and cellular cofactors become attached to the transcription complex during its transit through TAR.

Human immunodeficiency virus type 1 and 2 Tat proteins specifically interact with RNA polymerase II.

The Tat-responsive region (TAR) element is a critical RNA regulatory element in the human immunodeficiency virus (HIV) long terminal repeat, which is required for activation of gene expression by the transactivator protein Tat. Recently, we demonstrated by gel-retardation analysis that RNA polymerase II binds to TAR RNA and that Tat prevents this binding even when Tat does not bind to TAR RNA. These results suggested that direct interactions between Tat and RNA polymerase II may prevent RNA polymerase II pausing and lead to Tat-mediated increases in transcriptional elongation. To test this possibility, we performed protein interaction studies with RNA polymerase II and both the HIV-1 and the closely related HIV-2 Tat protein. These studies indicated that both the HIV-1 and HIV-2 Tat proteins could specifically interact with RNA polymerase II. Mutagenesis of both HIV-1 and HIV-2 Tat demonstrated that the basic domains of both the HIV-1 and HIV-2 Tat proteins were required for this interaction. Furthermore, "far Western" analysis suggested that the largest subunit of RNA polymerase II was the site for interaction with Tat. The interactions between Tat and RNA polymerase II were of similar magnitude to those detected between RNA polymerase II and the cellular transcription factor RAP30, which stably associates with RNA polymerase II during transcriptional elongation. These studies are consistent with the model that RNA polymerase II is a cellular target for Tat resulting in Tat-mediated increases in transcriptional elongation from the HIV long terminal repeat.

Parvovirus B19 promoter at map unit 6 confers autonomous replication competence and erythroid specificity to adeno-associated virus 2 in primary human hematopoietic progenitor cells.

The pathogenic human parvovirus B19 is an autonomously replicating virus with a remarkable tropism for human erythroid progenitor cells. Although the target cell specificity for B19 infection has been suggested to be mediated by the erythrocyte P-antigen receptor (globoside), a number of nonerythroid cells that express this receptor are nonpermissive for B19 replication. To directly test the role of expression from the B19 promoter at map unit 6 (B19p6) in the erythroid cell specificity of B19, we constructed a recombinant adeno-associated virus 2 (AAV), in which the authentic AAV promoter at map unit 5 (AAVp5) was replaced by the B19p6 promoter. Although the wild-type (wt) AAV requires a helper virus for its optimal replication, we hypothesized that inserting the B19p6 promoter in a recombinant AAV would permit autonomous viral replication, but only in erythroid progenitor cells. In this report, we provide evidence that the B19p6 promoter is necessary and sufficient to impart autonomous replication competence and erythroid specificity to AAV in primary human hematopoietic progenitor cells. Thus, expression from the B19p6 promoter plays an important role in post-P-antigen receptor erythroid-cell specificity of parvovirus B19. The AAV-B19 hybrid vector system may also prove to be useful in potential gene therapy of human hemoglobinopathies.

Oligomerization of CD4 is required for stable binding to class II major histocompatibility complex proteins but not for interaction with human immunodeficiency virus gp120.

Previous studies have failed to detect an interaction between monomeric soluble CD4 (sCD4) and class II major histocompatibility complex (MHC) proteins, suggesting that oligomerization of CD4 on the cell surface may be required to form a stable class II MHC binding site. To test this possibility, we transfected the F43I CD4 mutant, which is incapable of binding to class II MHC or human immunodeficiency virus (HIV) gp120, into COS-7 cells together with wild-type CD4 (wtCD4). Expression of F43I results in a dominant negative effect: no class II MHC binding is observed even though wtCD4 expression is preserved. Apparently, F43I associates with wtCD4 oligomers and interferes with the formation of functional class II MHC binding structures. In contrast, F43I does not affect the binding of gp120 to wtCD4, implying that gp120 binds to a CD4 monomer. By production and characterization of chimeric CD4 molecules, we show that domains 3 and/or 4 appear to be involved in oligomerization. Several models of the CD4-class II MHC interaction are offered, including the possibility that one or two CD4 molecules initially interact with class II MHC dimers and further associate to create larger complexes important for facilitating T-cell receptor crosslinking.

CT-2576, an inhibitor of phospholipid signaling, suppresses constitutive and induced expression of human immunodeficiency virus.

Viruses such as human immunodeficiency virus (HIV) require cellular activation for expression. Cellular activation in lymphoid cells is associated with augmented accumulation of certain phosphatidic acid (PA) species derived from the hydrolysis of glycan phosphatidylinositol (GPI). This suggests that activation of a phospholipid pathway may play a role in initiation of viral replication. To test this hypothesis, we examined the effect of tat gene expression on the production of cellular PA species, as the Tat protein is essential for HIV expression and has been implicated in activating the expression of multiple host cellular genes. Expression of tat increased the expression of PA. We then tested whether synthetic inhibitors of PA metabolism would inhibit activation of the HIV long terminal repeat by Tat and tumor necrosis factor alpha (TNF-alpha). CT-2576 suppressed both PA generation induced by Tat and HIV long terminal repeat-directed gene expression in response to Tat or TNF-alpha at a posttranscriptional step. CT-2576 also inhibited constitutive as well as TNF-alpha- and interleukin 6-induced expression of HIV p24 antigen in chronically infected U1 cells and in peripheral blood lymphocytes acutely infected with a clinical isolate of HIV. Pharmacological inhibition of synthesis of selected PA species may therefore provide a therapeutic approach to suppression of HIV replication.

Análise de ligação do gene de resistência Zym-2 com marcadores microssatélites e reação de acessos de meloeiro ao Zucchini yellow mosaic virus (ZYMV)

As viroses causam perdas significativas na cultura do melão. Dentre essas, o vírus do mosaico amarelo da abobrinha-de-moita (Zucchini yellow mosaic virus- ZYMV) possui grande importância para a cultura e é encontrado em todos os locais de plantio de cucurbitáceas. O controle desse vírus através da resistência genética é a forma mais eficiente de manejo. O acesso PI414723 é a única fonte de resistência de meloeiro ao ZYMV. Essa resistência é oligogênica e supostamente condicionada por três genes dominantes: Zym-1, Zym-2 e Zym-3. A localização cromossômica do gene Zym-1 já foi confirmada no grupo de ligação 2, próximo ao marcador CMAG36. Entretanto, a localização de Zym-2 ainda carece de confirmação experimental, muito embora existam evidências de sua localização no grupo de ligação 10 (LGX). Sendo assim, um dos objetivos do presente trabalho foi confirmar a localização do gene Zym-2 através de análises de ligação com marcadores microssatélites (SSRs). Para tanto, foi utilizada uma população F2 derivada do cruzamento PI414723 x \'Védrantais\'. As plantas foram inoculadas mecanicamente com o isolado RN6-F, patótipo 0, duas vezes em um intervalo de 24 h. A confirmação da infecção e a quantificação dos títulos virais nas plantas F2 foram realizadas através do teste PTA-ELISA. O DNA genômico das plantas foi extraído da primeira folha verdadeira e utilizado nas reações de PCR com primers específicos para SSRs selecionados pertencentes ao LGX. Observou-se uma distribuição assimétrica de classes de absorbância e maior frequência de indivíduos F2 na classe com menor valor (0,1 a 0,2), sugerindo a existência de um gene de efeito maior. O teste chi-quadrado mostrou que todos os marcadores segregaram na frequência esperada (1:2:1), exceto o marcador CMCT134b. A ligação do Zym-2 aos marcadores foi confirmada por meio de regressão linear simples. Dos marcadores analisados, a regressão linear foi significativa para MU6549 e CMBR55, com p-valores de 0,011 e 0,0054, respectivamente. As análises de ligação mostraram que as ordens e as distâncias entre os marcadores condizem com os mapas presentes na literatura. Um segundo objetivo do estudo foi o de avaliar a reação ao ZYMV de 42 acessos de meloeiro oriundos da região Nordeste do Brasil, com o intuito de explorar novas fontes de resistência. Foram realizados dois experimentos utilizando a mesma metodologia citada anteriormente. O título viral médio entre os acessos variou de 0,123 a 0,621 no experimento 1 e de 0,019 a 0,368 no experimento 2. Alguns acessos apresentaram consistentemente baixos títulos virais, próximos aos do acesso resistente PI414723 e dos controles negativos (plantas não inoculadas da cultivar \'Védrantais\'). Portanto, estes acessos mostram-se como potenciais fontes de resistência ao vírus para o emprego em programas de melhoramento.