Jerry P. Weir

Regulation of herpes simplex virus gene expression

Expression of the more than 80 individual genes of herpes simplex virus 1 (HSV-1) takes place in a tightly regulated sequential manner that was first described over 20 years ago. Investigations since that time have focused on understanding the mechanisms that regulate this orderly and efficient expression of viral genes. This review examines recent findings that have shed light on how this process is regulated during productive infection of the cell. Although the story is still not complete, several aspects of HSV gene expression are now clearer as a result of these findings. In particular, several new functions have recently been ascribed to some of the known viral regulatory proteins. The results indicate that the viral gene expression is regulated through transcriptional as well as post-transcriptional mechanisms. In addition, it has become increasingly clear that the virus has evolved specific functions to interact with the host cell in order to divert and redirect critical host functions for its own needs. Understanding the interactions of HSV and the host cell during infection will be essential for a complete understanding of how viral gene expression is regulated. Future challenges in the field will be to develop a complete understanding of the mechanisms that temporally regulate virus gene expression, and to identify and characterize the relevant interactions between the virus and the distinctive cell types normally infected by the virus.

Reproducibility of Serologic Assays for Influenza Virus A (H5N1)

Results for clade 1 viruses were more consistent among laboratories when a standard antibody was used.

FDA/NIH/WHO public workshop on immune correlates of protection against influenza A viruses in support of pandemic vaccine development, Bethesda, Maryland, US, December 10-11, 2007.

The goals of the workshop were to identify gaps in our knowledge and abilities to address the unique challenges encountered in the development of vaccines intended to protect against pandemic influenza and to facilitate implementation of a global research agenda to improve efficacy assessment of pandemic influenza vaccines. This workshop included discussions on: (i) current knowledge regarding immune correlates of protection against seasonal influenza; (ii) human immune responses to avian influenza infection and vaccines for novel influenza viruses; (iii) limitations of currently available assays to evaluate vaccine immunogenicity; and (iv) potential insights from animal models for correlates of protection against avian influenza.

Smallpox Vaccine Does Not Protect Macaques with AIDS from a Lethal Monkeypox Virus Challenge

It is unknown whether smallpox vaccination would protect human immunodeficiency virus type 1 (HIV-1)-infected individuals, because helper CD4(+) cells, the targets of HIV-1 infection, are necessary for the induction of both adaptive CD8(+) cell and B cell responses. We have addressed this question in macaques and have demonstrated that, although smallpox vaccination is safe in immunodeficient macaques when it is preceded by immunization with highly attenuated vaccinia strains, the macaques were not protected against lethal monkeypox virus challenge if their CD4(+) cell count was <300 cells/mm(3). The lack of protection appeared to be associated with a defect in vaccinia-specific immunoglobulin (Ig) switching from IgM to IgG. Thus, vaccination strategies that bypass CD4(+) cell help are needed to elicit IgG antibodies with high affinity and adequate tissue distribution and to restore protection against smallpox in severely immunocompromised individuals.

Prime-boost immunization with DNA and modified vaccinia virus Ankara vectors expressing herpes simplex virus-2 glycoprotein D elicits greater specific antibody and cytokine responses than DNA vaccine alone

Several reports have indicated that prime-boost strategies of vaccination can enhance the level of specific immunity induced by nucleic acid vaccines. The present report describes such a strategy with herpes simplex virus (HSV)-2 glycoprotein D (gD), using combinations of plasmid vector that expresses gD (pgD2) and a recombinant modified vaccinia virus Ankara vector that expresses gD (MVA-gD2). The IgG antibody response to gD and the HSV-2 neutralizing antibody response were greatest when the MVA-gD2 vector was used as the priming immunization and then was boosted with either pgD2 or MVA-gD2. Determination of the isotype profile of MVA-gD2-primed mice revealed a much broader distribution of isotypes than that seen after DNA vaccination. In addition, antigen-stimulated spleen cells from mice primed with MVA-gD2 and boosted with either MVA-gD2 or pgD2 produced higher levels of interleukin-2 and interferon-gamma than did those from pgD2-primed mice, indicating that a prime-boost immunization strategy that uses the MVA and plasmid DNA vector dramatically enhances and diversifies the humoral and cellular immune response to HSV-2 gD.

Recombinant vaccinia virus expressing the herpes simplex virus type 1 glycoprotein C protects mice against herpes simplex virus challenge

The gene encoding the herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) was isolated and cloned into a vaccinia virus insertion vector, and the resulting vaccinia-gC vector was used to construct a recombinant vaccinia virus that expressed gC (VVgC5). Infection of cells with VVgC5 resulted in cell surface expression of authentic HSV-1 gC. HSV-1 gC-specific neutralizing antibodies were produced in VVgC5-immunized mice, and lymphocytes exhibited an HSV-1-specific proliferation response in vitro following infection. More importantly, VVgC5-immunized mice were resistant to subsequent lethal HSV-1 challenge.

Antibody Response and Protective Capacity of Plasmid Vaccines Expressing Three Different Herpes Simplex Virus Glycoproteins

Plasmid expression vectors were constructed that contained the genes encoding herpes simplex virus 1 (HSV-1) glycoproteins C (gC), D (gD), and E (gE). Mice receiving two intramuscular injections of expression plasmid (50 microg) produced a specific HSV-1 antibody response. Mice receiving the gD plasmid were protected against a lethal intraperitoneal challenge of HSV-1 (5 x 10(4) pfu) but not against more demanding challenge doses. Protection with gC or gE plasmid vaccination could be demonstrated only if the inoculating dose of DNA was increased to 250 microg. In contrast, all mice immunized with vaccinia recombinants expressing either gC or gE survived HSV-1 challenge. Analysis of the HSV-1 antibody isotype produced by plasmid immunization revealed a response dominated by IgG2a. Combination delivery of all three glycoprotein expression plasmids provided better protection against lethal challenge, but mice receiving the combination were still not able to withstand increased challenge doses of virus.

Protective immunity against herpes simplex virus generated by DNA vaccination compared to natural infection

To evaluate the utility of plasmid DNA vaccination against disease caused by herpes simplex virus (HSV), we compared the strength of protection against lethal challenge following natural virus infection with that following vaccination with a plasmid encoding HSV glycoprotein gD (gD-DNA). We further determined the cellular basis of each type of protection using lymphocyte deficient knockout mice. Establishment of immunity to HSV using live virus immunization required CD8+ T cells and B cells, but not CD4+ or gamma/delta+ T cells, and was related to specific antibody levels; surprisingly, CD4 knockout mice had large quantities of IgG anti-HSV serum antibodies. Establishment of immunity to HSV using gD-DNA immunization approached the strength of that generated following sublethal infection, but was dependent on alpha/beta+ CD4+ T cells, CD8+ T cells, B cells, and even partially on gamma/delta+ T cells, and not strictly correlated with antibody levels.

An alternative method for preparation of pandemic influenza strain-specific antibody for vaccine potency determination

The traditional assay used to measure potency of inactivated influenza vaccines is a single-radial immunodiffusion (SRID) assay that utilizes an influenza strain-specific antibody to measure the content of virus hemagglutinin (HA) in the vaccine in comparison to a homologous HA reference antigen. Since timely preparation of potency reagents by regulatory authorities is challenging and always a potential bottleneck in influenza vaccine production, it is extremely important that additional approaches for reagent development be available, particularly in the event of an emerging pandemic influenza virus. An alternative method for preparation of strain-specific antibody that can be used for SRID potency assay is described. The approach does not require the presence or purification of influenza virus, and furthermore, is not limited by the success of the traditional technique of bromelain digestion and purification of virus HA. Multiple mammalian expression vectors, including plasmid and modified vaccinia virus Ankara (MVA) vectors expressing the HAs of two H5N1 influenza viruses and the HA of the recently emerging pandemic H1N1 (2009) virus, were developed. An immunization scheme was designed for the sequential immunization of animals by direct vector injection followed by protein booster immunization using influenza HA produced in vitro from MVA vector infection of cells in culture. Each HA antibody was highly specific as shown by hemagglutination inhibition assay and the ability to serve as a capture antibody in ELISA. Importantly, each H5N1 antibody and the pandemic H1N1 (2009) antibody preparation were suitable for use in SRID assays for determining the potency of pandemic influenza virus vaccines. The results demonstrate a feasible approach for addressing one of the potential bottlenecks in inactivated pandemic influenza vaccine production and are particularly important in light of the difficulties in preparation of potency reagent antibody for pandemic H1N1 (2009) virus vaccines.

Genomic Organization and Evolution of the Human Herpesviruses

Members of the Herpesviridae family have been isolated from most animal species examined. Of approximately 100 individual virus species, eight have been isolated from humans, and three of these only within the last 10 years. Fortunately, there is now an enormous amount of sequence data from many of these viruses, particularly the eight human herpesviruses. This wealth of sequence information from such a diverse group of related viruses provides a unique resource for studies of viral gene evolution, comparative gene function, and virus identification.