An optimally effective AIDS vaccine would likely require the induction of both neutralizing antibody and cell-mediated immune responses, which has proven difficult to obtain in previous clinical trials. Here we report on the induction of human immunodeficiency virus type-1 (HIV-1)-specific immune responses in healthy adult volunteers that received the multi-gene, polyvalent, DNA prime–protein boost HIV-1 vaccine formulation, DP6-001, in a Phase I clinical trial. Robust cross-subtype HIV-1-specific T cell responses were detected in IFN-γ ELISPOT assays. Furthermore, we detected high titer serum antibody responses that recognized a wide range of primary HIV-1 Env antigens and also neutralized pseudotyped viruses that express the primary Env antigens from multiple HIV-1 subtypes. These findings demonstrate that the DNA prime–protein boost approach is an effective immunization method to elicit both humoral and cell-mediated immune responses in humans, and that a polyvalent Env formulation could generate broad immune responses against HIV-1 viruses with diverse genetic backgrounds.

Mouse hepatitis virus (MHV) is the prototype of group II coronaviruses and one of the most extensively studied coronaviruses. Here, we describe a reverse genetic system for MHV (strain A59) based upon the cloning of a full-length genomic cDNA in vaccinia virus. We show that the recombinant virus generated from cloned cDNA replicates to the same titers as the parental virus in cell culture (ϳ10 9 PFU/ml), has the same plaque morphology, and produces the same amounts and proportions of genomic and subgenomic mRNAs in virusinfected cells. In a mouse model of neurological infection, the recombinant and parental viruses are equally virulent, they replicate to the same titers in brain and liver, and they induce similar patterns of acute hepatitis, acute meningoencephalitis, and chronic demyelination. We also describe improvements in the use of the coronavirus reverse genetic system based on vaccinia virus cloning vectors. These modifications facilitate (i) the mutagenesis of cloned cDNA by using vaccinia virus-mediated homologous recombination and (ii) the rescue of recombinant coronaviruses by using a stable nucleocapsid protein-expressing cell line for the electroporation of infectious full-length genomes. Thus, our system represents a versatile and universal tool to study all aspects of MHV molecular biology and pathogenesis. We expect this system to provide valuable insights into the replication of group II coronaviruses that may lead to the development of novel strategies against coronavirus infections, including the related severe acute respiratory syndrome coronavirus.

An optimally effective AIDS vaccine would likely require the induction of both neutralizing antibody and cell-mediated immune responses, which has proven difficult to obtain in previous clinical trials. Here we report the induction of Human Immunodeficiency Virus Type-1 (HIV-1)-specific immune responses in healthy adult volunteers that received the multi-gene, polyvalent, DNA prime-protein boost HIV-1 vaccine formulation, DP6−001 in a Phase I clinical trial conducted in healthy adult volunteers of both genders. Robust cross-subtype HIV-1-specific T cell responses were detected in IFNγ ELISPOT assays. Furthermore, we detected high titer serum antibody responses that recognized a wide range of primary HIV-1 Env antigens and also neutralized pseudotyped viruses that express the primary Env antigens from multiple HIV-1 subtypes. These findings demonstrate that the DNA prime-protein boost approach is an effective immunization method to elicit both humoral and cellmediated immune responses in humans, and that a polyvalent Env formulation could generate broad immune responses against HIV-1 viruses with diverse genetic backgrounds.

The coding capacity of human cytomegalovirus (HCMV) for glycoproteins by far exceeds that of other herpesviruses. Few of these proteins have been characterized so far. We have investigated the gene product of reading frame UL132. The putative protein product of UL132 is a glycoprotein with a theoretical mass of 29.8 kDa. Transcription analysis revealed that the gene is transcribed with a true late kinetics from the laboratoryadapted strain AD169 and the low-passage isolate TB40E. Two proteins of 22 to 28 kDa and 45 to 60 kDa were detected in virus-infected cells as well as in extracellular virions. The larger protein carried N-linked carbohydrates. Both protein forms were present in laboratory-adapted strains as well as in low-passage isolates of HCMV. Recombinant viruses with the UL132 gene deleted were constructed in the low-passage HCMV isolate PAN as well as the high-passage isolate AD169. Deletion of UL132 from either genome resulted in a pronounced replication deficit with a reduction of approximately 100-fold for HCMV strain AD169. Thus, the protein product of the UL132 reading frame represents a structural viral glycoprotein of HCMV that has an important function for viral replication in tissue culture.

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