Herpes simplex virus (HSV) infections have been recognized since ancient Greek times. Prevention of human HSV disease by vaccines has been attempted for nearly two centuries without apparent success, especially during most of the 20th century. Indeed, the development of an efficacious HSV vaccine is very much needed. In the United States alone, more than 100 million individuals are infected by HSV-I and at least 40 to 60 million individuals by HSV-2.1 Overt clinical recurrences may only be apparent in approximately 20% to 30% of individuals infected by HSV, an approximation at best. Regardless, there is a reservoir of individuals who have been infected with HSV and may intermittently shed virus in the absence of symptoms.
As the end of the century approaches and HSV vaccine trials gain urgency in the clinical research community, fundamental questions that must be asked include: What should a vaccine against HSV accomplish? To whom should it be administered? Should it prevent or, alternatively, just ameliorate primary infection? Should an HSV vaccine be expected to prevent horizontal transmission of infection between sexual partners or vertical transmission from mother to baby? If so, how effective must it be? Can it be used to treat frequently recurrent HSV infections - either labial or genital? Will subunit vaccines provide lasting immunity? Will adjuvant technology elevate the potential of subunit vaccines without undue reactogenicity? If we administer live attenuated vaceines, will the property of establishment of latency be viewed as an undesirable characteristic of the vaccine?
In the end, HSV vaccinologists must define those qualities of host immunity that equate with protection. The answers to these questions, as well as a myriad of others, await definition in clinical trials. However, HSV vaccine research efforts will strive to develop a vaccine that induces such immunity, both humoral and cell-mediated, in the recipient that infection can be prevented - indeed, a lofty goal. By preventing the primary infection, an ideal HSV vaccine would also prevent the colonization of sensory ganglia, leaving no latent source of virus for subsequent recurrences. Thus, it is open to discussion whether the criteria of success for candidate vaccines should be one or a combination of the following effects:
* complete abrogation or amelioration of primary clinical episodes,
* prevention of the colonization of the ganglia,
* suppression or reduction in the frequency and/or severity of recurrences,
* reduction of the transmission (duration and/or quantity) of the virus during primary and/or recurrent episodes,
* reduction of asymptomatic transmission (frequency, duration, quantity).
These issues must be weighed in the context of the age of the target population and the duration of the desired results. Regardless, this article summarizes the status of these issues and offers hope for amelioration, if not prevention, of human HSV disease.
HUMAN HERPES SIMPLEX VIRUS VACCINE DEVELOPMENT
Insight into the problems of HSV vaccine development was acquired early in natural history and vaccine studies. Such observations from these early vaccine efforts included the appearance of lesions at the vaccine site and the development of recurrent lesions following either natural disease or immunization. Neutralizing antibodies to HSV were found in the serum of previously infected adults some of whom subsequently developed recurrent lesions, albeit less severe than those associated with the initial episode.2 This observation codifies the unique biologic property of HSV: namely its ability to recur in the presence of humoral immunity - a characteristic known as reactivation of latent infection. Only individuals with neutralizing antibodies developed recurrent lesions, a paradoxical finding given the classical lessons of such infectious diseases as measles and rubella whereby antibodies provided protection from subsequent disease.3
Herpes simplex virus vaccine efforts have been directed toward the prevention of recurrent infections, using live but attenuated viruses. All attempts to prevent recurrences by immunizations in humans with these live viruses have either (ailed or have been only partially effective. In general, four approaches have evaluated vaccines in humans as either therapeutics or preventatives, including use of: 1} wild-type virus, 2) inactivated (or killed) virus, 3) subunit vaccines, and 4) live vaccines. Each will be discussed separately.
Numerous clinical investigators have attempted to alter the pattern of recurrences by inoculation of: 1) autologous virus, 2) virus from another infected individual, or 3) in one set of experiments, virus recovered from an experimentally infected rabbit. In each circumstance, the inoculation of virus led to evidence of infection at the site of injection in as many as 40% to 80% of volunteers.4 Efficacy has been reported in limited numbers of volunteers but without the use of matched unvaccinated controls. In some of these cases, inoculation led to recurrences of latent virus. In large part, live viruses were abandoned as immunogens on the grounds that many subjects failed to develop lesions at the site of inoculation and, therefore, it was perceived that the patient did not have an "adequate take." The analogy was the requirement of a cutaneous take following smallpox immunization. Today, inoculation of either autologous or heterologous virus is unacceptable.
Inactivated (or Killed) Virus
Killed HSV vaccines have been studied in animal models, often with good results.5·6 When these vaccines are administered to patients in order to alleviate recurrences, most studies failed to include unvaccinated controls. Thus, significant bias is introduced because volunteers in other vaccine trials have experienced 30% to 70% decrease in the frequency and severity of recurrent lesions, simply from having received placebo.
The initial inactivated vaccines were made from phenol-treated tissues obtained from infected animals. Because of the potential for demyelination following the administration of animal proteins, these vaccines attracted little biomédical attention. Subsequently, ultraviolet light inactivation of purified vims derived from tissue cultures provided greater impetus for the vaccine field. Numerous reports have suggested both successes and failures of such approaches. Viral antigens obtained from amniotic or allantoic fluid, chorioallantoic membranes, chick-cell cultures, sheep-kidney cells, and rabbit-kidney cells inactivated either by formalin, ultraviolet light, or heat led to a series of vaccine studies in thousands of volunteers. With one exception, each of these studies reported significant improvement in as many as 60% to 80% of subjects.
From these studies, several important observations were made. First, despite repeated inoculations, antibody titers (as measured by neutralization or complement fixation) remained unchanged in the majority of humans or demonstrated only slight increases. Second, while vaccines reported few side effects, some investigators suggested that in volunteers with keratitis autoimmune phenomena might make herpetic disease worse. Third, there were few placebocontrolled studies of inactivated vaccines for recurrent disease. The results of these studies are discrepant, even when the same vaccine was used. The conclusion from these investigations was that there may be some initial, vaccine-induced benefits for humans with recurrent infection; however, long-term benefit could not be established.
Finally, in one prospective study of primary prevention, 10 children received vaccine and 10 received a placebo; nevertheless, HSV gingivostomatitis developed in an equal number of patients in both groups on long-term follow-up. The perception of the biomédical research community is that these vaccines do not meet accepted standards for Iicensure.
Subunit vaccines, which use only a portion of the organism to provide the antigenic stimulus, evolved from attempts to: 1) remove viral DNA, thereby eliminating the potential for either cellular transformation or establishment of latency; 2) enhance antigenic concentration and include stronger immune responses; and 3) exclude any possibility of contamination with residual live virus. Crude subunit vaccines have been prepared by combining antigen extraction from infected cell lysates by detergent and subsequent purification. One such vaccine was studied in sexual partners of individuals known to have genital herpes; the number of subjects developing herpetic infection was nearly equal between placebo and vaccine recipients, contradicting any value in using the proposed vaccine.
More sophisticated subunit vaccines have evolved from this era of molecular biology such as those derived from cloning of specific glycoproteins in either yeast or Chinese hamster ovary (CHO) cells,7 as well as by other methods. By cloning, the glycoprotein B-2 (gB-2) and the glycoprotein D-2 (gD-2) genes have been truncated to yield immunogenic proteins in CHO cells. Glycoproteins B and D of HSV-2 were selected because they are essential for viral replication and contribute significantly to viral infectivity. These subunit vaccines, in combination with a variety of adjuvants, and others have been studied in animal models including mice, guinea pigs, and rabbits. Neutralizing antibodies can be detected, but in varying amounts. The quantity of neutralizing antibodies appeared to correlate with the degree of protection upon challenge. Each used different routes of challenge such as skin, lip abrasion, intravaginal, ear pinna, foot pad, intraperitoneal, or ocular.
Despite conflicting studies, subunit vaccines elicit a degree of protection as evidenced by amelioration of morbidity and reduction in mortality in the immunized animals. However, several injections with an adjuvant are required for protection. Protection of rodents is significantly easier than in higher primate species. Vaccination of subhuman primates results in the production of neutralizing antibodies. Amnestic responses can be elicited following re-immunization. The degree of protection is unclear.
Evaluation of gB and/or gD recombinant vaccines in humans are in progress, with extensive Phase I and II gD-2 vaccine experience. Vaccination with the gD-2 construct results in an increase in the geometric mean antibody titer to gD-2; however, lymphocyte blastogenic responses are not consistent among populations. Either 30 µg or 100 µg of purified gD-2 was used as die immunogen with alum. More recently, the use of a squalene adjuvant (MF-59) appears to enhance immunogenicity.8 This vaccine has the potential of amelioration, if not prevention, of primary disease and may benefit those with frequent recurrences. Data presented from a recent clinical trial have suggested a 30% decrease in recurrences and shedding in individuals with frequently recurrent genital HSV infection (Straus, personal communication, 1993). A construct of gB~2 in combination with gD-2 and MF-59 is being evaluated for treatment of recurrent genital herpes and for prevention of primary infection in HSV-2 discordant monogamous sexual partners.
Other vaccine constructs include gD combined with Upophilic muramyl tripeptide (MTP-PE), which affords a high level of protection from HSV disease in the animal model. The quantity of antigen in the vaccine results in varying host immune responses. The quantity of neutralizing antibody elicited by immunization and the total HSV antibody titer (as measured by ELÌSA) are higher after vaccination than following natural infection, and these levels correlated with protection from disease.
The expression of gD using recombinant DNA technology to create Escherichia coli or bacteriophage M13 as vectors has been reported. In addition, gD-2 of HSV-I has been expressed in CHO and Saccharomyces cerevisiae cells.9
Live vaccines have been considered preferable for decades because they are likely to provide a high level of protection, as has been documented with numerous viral pathogens such as polio, measles, mumps, and rubella. The rationale for the selection of live viral vaccines is that, because they replicate in the recipient, the resulting immunity should be longer lasting. Moreover, these vaccines usually require smaller quantities of antigen and, therefore, should be less expensive. Several approaches for producing live virus vaccines have been attempted. These include: HSV mutants, heterologous herpesviruses, antigens expressed in non-HSV viral vectors, and genetically engineered viruses.
HS V Mutants. There is a wide range in virulence, and conversely of attenuation, among wild-type HSV isolates. Conceivably, an avirulent strain of HSV could be used to a vaccine, but reversion from nonpathogenic to pathogenic strains can occur by serial passage in animal hosts. This possibility creates such concern that the use of such mutants for vaccination is unacceptable.
Heterologous (Nonhuman) Herpesvirus Vaccines. While considered for nonhutnan herpesvirus infections (eg, Marek's disease, a lymphoproliferative disease of chickens that produces lymphomas), the use of heterologous herpesviruses as a human vaccine is considered untenable.
Antigens Expressed in Live, Non-HSV Vectors. Vaccinia virus has been proposed as a vector for delivering antigens to animals and humans.10 The principle of inserting foreign genes into vaccinia has been exploited for the expression of the gB and gD genes of HSV. Significant concern has been raised over the use of vaccinia for this purpose. Mainly because of the occurrence in the past of vaccinia gangrenosum and disseminated vaccinia in individuals who were vaccinated to prevent smallpox. Furthermore, immune memory in individuals previously immunized against smallpox may prevent recognition of any foreign insert, as supported by animal model data. Avian pox virus has been considered an alternative, as have adenoviruses.
Genetically Engineered HSV. Molecular biology makes it possible to modify the genome of large DNA viruses and construct genetically engineered, attenuated live viruses,11 including HSV. These viruses were engineered to be attenuated, protect against HSV-I or -2 infections, provide markers of immunization distinct from wild-type infections, and serve as vectors to express other immunogens.
The construction of live-attenuated HSV was based on the use of an HSV-1 (HSV-I[F]) as a backbone to avoid the presence of the sequences of HSV-2 reported to be associated with transformation in cell cultures. The genome was deleted in the domain of the viral thymidine kinase (TK) gene and in the junction region of the U1 and U8 segments in order to excise some of the genetic loci responsible for neurovirulence, as well as to create convenient sites and space within the genomes for insertion of other genes. Finally, an HSV-2 DNA fragment encoding the HSV-2 glycoproteins D, G, and I were inserted in place of the interval inverted repeat. The purpose of type 1 genes was to broaden the spectrum of the immune response and to create a chimeric pattern of antibody specificities as a serological marker of vaccination. The resulting recombinant, designed as R701 7, had no TK activity and, therefore, would be resistant to acyclovir. Therefore, another recombinant was created, designated R7020, by insertion of the TK gene next to the HSV-2 DNA fragment. Since this virus expresses HSV-TK, it is susceptible to acyclovir. When analyzed by restriction enzyme digestion, the DNA of these recombinants shows patterns that enable their unambiguous identification.
When evaluated in rodent models, the two constructs are considerably attenuated in their pathogenicity and ability to establish latency, and were capable of inducing protective immunity. The recombinants did not regain virulence, nor did they change DNA restriction enzyme cleavage patterns when subjected to serial passages in the mouse brain.12 Remarkably, the TK virus R7017 behaved similarly to the TK + virus R7020.
These results were corroborated by studies in owl monkeys (Aotus trivirgatus).13 While 100 plaqueforming units (PFUs) of wild-type viruses administered by peripheral routes were ratal to the monkeys, recombinants given by various routes in amounts at least 105-fold greater were innocuous or produced mild infections, even in the presence of immunosuppression by total lymphoid irradiation.
Human studies with this vaccine indicate that neutralizing antibodies can be generated with either one or two doses of vaccine administered at a dosage of IO5 PFU.14 However, because replication of this vaccine in MRC-5 cells is limited, alternative constructs are being developed that have an enhanced capacity to replicate. One of these constructs involves the deletion of the gamma-1 34-5 gene of which there are two copies in the inverted repeats of the unique long sequence. These constructs are under evaluation in animal models.15
Within the last several years a very focused effort on developing vaccines to prevent HSV infections has led to several candidates. The lessons from past failures will have direct applicability to the studies currently underway. We have learned that seronegative individuals at high-risk for infection represent ideal candidates for participation in vaccine trials, while individuals with frequent recurrences probably do not offer the opportunity for absolute protection. As a consequence, vaccination should be scheduled for a time prior to exposure to HSV. For a vaccine designed to prevent HSV-2 infections, this would be early in adolescence prior to the onset of sexual activity.
Current efficacy studies will have to be doubleblind and placebo'controlled. Attention will have to be directed to the number of volunteers required for appropriate statistical analyses in order to comply wich proper trial designs. After enrollment into a prospective clinical trial, the diversity of clinical HSV diseases and the lack of predictability of patterns of recurrence will mandate a very careful prospective evaluation for both symptomatic and asymptomatic evidence of infection in vaccine recipients. On both clinical and laboratory levels, detailed evaluations will have to determine presence or absence of subsequent wild-type infection.
The intellectual and scientific challenges posed by the development of a vaccine to prevent HSV infections are extremely rewarding. Hopefully, within the next several years excellent clinical trials will help establish the value of such approaches.
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