Comparison of different forms of herpes simplex replication-defective mutant viruses as vaccines in a mouse model of HSV-2 genital infection.

Some subunit vaccines composed of herpes simplex virus (HSV) glycoproteins have been shown to protect guinea pigs against primary and recurrent genital infection by HSV-2. However, these vaccines were ineffective or only marginally effective in clinical trials. To attempt to define an animal model that would better discriminate the protective capacity of different vaccine formulations, we have examined the requirements for vaccine-induced protection against HSV-2 infection and disease in a mouse genital model. Unlike the guinea pig model where inactivated viral vaccines can protect nearly as well as live viral vaccines, inactivated viral vaccine afforded little protection in this mouse model. Using replication-defective mutant viruses as a form of live viral vaccine, we found that the extent of protection conferred by live vaccine was proportional to the amount of replication-defective mutant virus inoculated, over doses from 10(4) to 10(6) PFU. Furthermore, the mouse genital model showed quantitative differences in the degree of protection induced by various viral vaccine constructs. An HSV-2 replication-defective mutant virus protected better than an HSV-1 replication-defective mutant that expressed HSV-2 glycoprotein D, which in turn protected better than an HSV-2 replication-defective mutant virus. We conclude that this mouse genital model can rank different vaccine constructs for their capacity to induce protective immunity. Thus, genital infection of the mouse with HSV-2 may provide a stringent animal model that can predict the relative capacity of viral vaccines to stimulate protective immunity against HSV-2.

Humoral response to herpes simplex virus is complement-dependent.

The complement system represents a cascade of serum proteins, which provide a major effector function in innate immunity. Recent studies have revealed that complement links innate and adaptive immunity via complement receptors CD21/CD35 in that it enhances the B cell memory response to noninfectious protein antigens introduced i.v. To examine the importance of complement for immune responses to virus infection in a peripheral tissue, we compared the B cell memory response of mice deficient in complement C3, C4, or CD21/CD35 with wild-type controls. We found that the deficient mice failed to generate a normal memory response, which is characterized by a reduction in IgG antibody and germinal centers. Thus, complement is important not only in the effector function of innate immunity but also in the stimulation of memory B cell responses to viral-infected cell antigens in both blood and peripheral tissues.

Immunization against genital herpes with a vaccine virus that has defects in productive and latent infection.

An effective vaccine for genital herpes has been difficult to achieve because of the limited efficacy of subunit vaccines and the safety concerns about live viruses. As an alternative approach, mutant herpes simplex virus strains that are replication-defective can induce protective immunity. To increase the level of safety and to prove that replication was not needed for immunization, we constructed a mutant herpes simplex virus 2 strain containing two deletion mutations, each of which eliminated viral replication. The double-mutant virus induces protective immunity that can reduce acute viral shedding and latent infection in a mouse genital model, but importantly, the double-mutant virus shows a phenotypic defect in latent infection. This herpes vaccine strain, which is immunogenic but has defects in both productive and latent infection, provides a paradigm for the design of vaccines and vaccine vectors for other sexually transmitted diseases, such as AIDS.

Influence of mucosal and parenteral immunization with a replication-defective mutant of HSV-2 on immune responses and protection from genital challenge.

Herpes simplex virus (HSV) most frequently initiates infection at a mucosal surface; thus mucosal immune responses are likely to be important in defense against HSV infection. We have examined the effects of eliciting mucosal as well as systemic immune responses on protection against genital challenge infection with virulent HSV-2 in mice immunized with a replication-defective mutant of HSV-2. In addition, we have examined the types of immune responses elicited by immunization by the different routes under conditions known to provide protection. We observed that immunizations at parenteral and distal mucosal sites generate immune responses that have an additive effect in protection against challenge infection with virulent HSV-2. Immunization at either of these sites alone prevented paralysis and death after challenge virus infection and reduced replication of the challenge virus in the genital mucosa, although subcutaneous immunization was more effective in reducing virus replication. Simultaneous immunization at the two sites led to the greatest reduction in mucosal replication of challenge virus. The type of response generated was also affected by the route of immunization. Subcutaneous immunization results in a strong systemic immune response that is somewhat biased toward a Th1 T cell response, while intranasal immunization induces mucosal as well as systemic immunity, as evidenced by HSV-specific IgA in vaginal secretions, and a stronger bias toward a Th1 response. These results suggest that mucosal immunization may complement protective immunity against HSV-2 genital infection generated by parenteral immunization with replication-defective mutant virus.

Mutations that render the promoter of the histidine operon of Salmonella typhimurium insensitive to nutrient-rich medium repression and amino acid downshift.

The effects of mutations in the promoter of the histidine operon of Salmonella typhimurium were examined in vivo. The wild-type chromosomal copy of the his promoter was replaced with mutations in the -10 hexamer sequence and in the region between the -10 hexamer and the transcriptional start point-termed the discriminator sequence. The substitutions were performed with a phage M13 allele replacement system. Expression of the his operon is known to correlate with levels of guanosine 5',3'-bispyrophosphate (ppGpp) in vivo. Strains containing either the wild-type his promoter or his promoter mutations were grown in both nutrient-rich and minimal media under steady-state conditions known to alter intracellular levels of ppGpp in a predictable way. The effect of the presence or absence of the his attenuator was assessed under these conditions as well. Expression of the his operon was studied by measuring the differential rate of beta-galactosidase synthesis with a his-lac transcriptional fusion. Regulation of the his operon in the promoter mutants was also studied under conditions of a transient amino acid downshift induced by the addition of serine hydroxamate to cultures growing in nutrient-rich medium. These growth conditions cause elevated levels of ppGpp. The results provide physiological confirmation of previous evidence obtained with a coupled transcription-translation system in vitro which indicated that ppGpp regulates interaction of RNA polymerase at the his promoter. More specifically, the in vivo evidence shows that the region of the his promoter that includes the -10 hexamer and discriminator sequences is the target at which ppGpp stimulates transcription.

Construction and characterization of a replication-defective herpes simplex virus 2 ICP8 mutant strain and its use in immunization studies in a guinea pig model of genital disease.

A replication-defective mutant of herpes simplex virus 2 (HSV-2) was engineered by replacing the ICP8 gene of HSV-2 strain 186 with an ICP8-lacZ fusion gene from the herpes simplex virus 1 (HSV-1) HD-2 mutant strain. The resulting virus, HSV-2 5BlacZ, is defective for growth in Vero cells but is capable of growth in a cell line that expresses HSV-1 ICP8. In Vero cells, the mutant virus is defective for DNA synthesis but is able to express many viral proteins at levels similar to those of wild-type virus, including several of the late kinetic class. SDS-PAGE and Western blot analysis demonstrated the expression of glycoproteins B and D by 5BlacZ in Vero cells. Initial studies have shown that immunization with 5BlacZ protects guinea pigs from intravaginal HSV-2 challenge. Immunized animals had less severe genital skin disease and reduced replication of the challenge virus in the genital tract during primary infection and reduced episodes of recurrent disease. Thus, HSV-2 ICP8 shows gene regulatory properties similar to those of HSV-1 ICP8, and this HSV-2 ICP8 mutant virus shows a phenotype similar to those of HSV-1 ICP8 mutant strains. Replication-defective mutants of HSV-2 offer a potential vaccine approach for immune intervention against HSV-2 genital disease and latent infection.