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Summary Objective To analyse the records of past influenza outbreaks to determine a definition for pandemics. Methods Analysis of publications of large outbreaks of influenza which have occurred since 1889/90, and to match the results against the current definitions of an influenza pandemic. Results According to the general understanding of a pandemic, nine outbreaks of influenza since 1889/90 satisfy the definition; however, for two of these, occurring in 1900 and 1933, the data are limited. The special condition for an influenza pandemic requires, in one definition, that the virus strain responsible could not have arisen from the previous circulating strain by mutation; and in the second, that the new strain be a different subtype to the previously circulating strain. Both these restrictions deny pandemic status to two, and possibly three, influenza outbreaks which were pandemics according to the more general understanding of the term. These observations suggest that a re-evaluation of the criteria which define influenza pandemics should be carried out. Conclusion The contradiction outlined above brings the previous definitions of an influenza pandemic into question; however, this can be resolved by defining an influenza pandemic by the following criteria. Thus, an influenza pandemic arises at a single, specific place and spreads rapidly to involve numerous countries. The haemagglutinin (HA) of the emergent virus does not cross-react serologically with the previously dominant virus strain(s), and there is a significant lack of immunity in the population against the emergent virus. These three criteria are interlinked and can be determined early to alert authorities who could respond appropriately. Other criteria associated with pandemics are necessarily retrospective, although important and valid. The implications of this definition are discussed.

Background: Chlamydia trachomatis is an important pathogen, being the commonest sexually transmitted bacterial disease in the Western world and is also implicated in a number of acute and chronic diseases. Persistent infections of C. trachomatis are particularly associated with chronic infections, which although eliciting an immune response, result in tissue damage leading to complications such as pelvic inflammatory disease. Interferon (IFN)-gamma is known to induce persistent infections of C. trachomatis both in vitro and in vivo. Methods: A model of IFN-gamma-induced persistence containing aberrant inclusions of C. trachomatis was developed in the HEp-2 cell line. Morphological changes to inclusions were assessed by fluorescence immunocytochemistry and transcript levels determined by Real-Time RT-PCR. To assess infectivity of C. trachomatis in an IFN-gamma-induced persistent state, cultures containing aberrant inclusions were inoculated onto fresh HEp-2 monolayers. Results: IFN-gamma induced aberrant inclusion formation at 0.01 ng/ml. Doses from 0.05 to 100 ng/ml did not significantly increase numbers of aberrant inclusions, and some normal inclusions were observed at the highest dose of IFN-gamma. Transfer of IFN-gamma-treated C. trachomatis onto fresh cultures confirmed the infectivity of these cultures. Real-Time RT-PCR identified apparent increased expression of the C. trachomatis heat-shock response genes ct604 and ct755 at 96-h post-infection. However comparisons with control cultures suggest that this more likely reflects a failure to down regulate gene expression as observed in untreated cultures. Conclusions: These data show that whereas IFN-gamma induces aberrant inclusion formation, many normal inclusions are still observed at high doses of IFN-gamma, and that the infectivity of such cultures is presumably from these. Transcriptional changes observed in response to IFN-gamma suggest a failure of the C. trachomatis life cycle in response to IFN-gamma, however IFN-gamma-induced transcriptional changes may be masked by the presence of normal inclusions. The implications of these observations in relation to models of persistence of C. trachomatis are discussed.

The aim of this study was to determine whether interleukin (IL)-6 and IL-8 concentrations, as well as numbers of seminal leukocytes in a population of infertile men, some of whom were Chlamydia trachomatis positive, were related to chlamydial infection. Our patient group included 255 men attending for diagnostic semen analysis as part of infertility investigations. Significantly raised levels of IL-8, but not IL-6, were found in C trachomatis-infected patients but not in uninfected patients. Raised IL-8 levels in semen were also associated with an increase in semen volume. There was a relationship between C trachomatis infection and lower progressive motile sperm, as well as an increase in seminal leukocytes. The overall prevalence rate for C trachomatis was 6.2%, and more infections were detected in semen than in first void urine. This study supports the suggestion that IL-8 might be used as a marker for male genital tract infection, especially when due to C trachomatis. In this study, there was a relationship between the presence of C trachomatis in semen and alterations of some semen parameters. Further investigations should be performed to understand the disparities of first void urine and semen testing for detection of C trachomatis in males.

The potential of a large number of commercial and experimental adjuvant preparations to enhance the immunogenicity of an HSV-1 glycoprotein subunit vaccine was investigated. Evaluation was based on toxicity, HSV-specific antibody production, and protection against lethal challenge. All adjuvants tested increased the titer of antigen specific Ig levels when compared to subunit vaccine alone. However, following challenge, a broad range of protective responses were noted. Statistically significant correlations were observed between IgG antibody levels post immunization and the observed protection and these were particularly associated with antibodies of the IgG2a and IgG2b subclasses. The results emphasize the requirement of adjuvants for vaccine formulation when using subunit preparations, and demonstrate that the magnitude and efficacy of the induced immune response varies greatly with the choice of adjuvant.

The effect of subunit vaccination on the incidence and severity of primary and recurrent genital herpes was investigated in the female guinea pig model of herpes simplex virus (HSV) type 2 genital infection. After prophylactic immunization with zwitterionic detergent-solubilized HSV-1 glycoproteins formulated with alhydrogel or as immunostimulating complex particles, significant reductions in the incidence and severity of primary herpetic illness were observed in both vaccinated groups compared with immunization-naive controls. There was a significant reduction in the incidence of spontaneous herpetic recurrences after administration of HSV-1 antigens formulated as immunostimulatory complexes to guinea pigs in a prophylactic mode (P

Therapeutic immunisation may represent a means of influencing viral infections that persist in the host by modulating the nature or level of host immunity. To assess the influence of the form of the antigenic stimulus on immunity to type-2 herpes simplex virus (HSV-2), mice pre-infected with sublethal doses of HSV-2 were immunised with various HSV-2 vaccine formulations prior to challenge infection with heterologous HSV-1. Measurements of interleukin-2 (IL-2), interleukin-4 (IL-4) and interferon-γ (IFN-γ) levels in mouse spleen cell cultures restimulated in vitro with HSV-2 antigens showed that, depending on the form of HSV-2 antigen preparation used in this therapeutic context, changes in the levels of these cytokines could be effected. Measurement of HSV-specific antibody by serological tests support the contention that immunisation of HSV-2-infected mice can either enhance the existing Th1-like immune response elicited following HSV-2 infection, or modulate this response towards a more Th2-like profile, and this is dependent on the form of the antigenic stimulus. The degree of protection against subsequent lethal, heterologous HSV-1 challenge infection varied according to the nature of the infection and the immunisation history of the animals.

The development of vaccines against the herpesviruses has major public health importance because of the wide spectrum of associated clinical disease with this virus in both immunocompetent and immunocompromised populations. Because these viruses establish latent infections capable of subsequent reactivation, both immunotherapeutic and prophylactic vaccine strategies are needed. A range of vaccine formulations has been devised, largely as a result of the rapid growth in knowledge in molecular microbiology and genetic engineering, including live and inactivated whole virus vaccines, and subunit vaccines consisting of recombinant viral glycoproteins in various adjuvants. The live attenuated virus Oka strain vaccine is now licensed for prophylaxis against varicella (VZV) in some countries. Recent studies with herpes simplex viruses (HSV) have demonstrated immunogenicity with glycoprotein vaccines; however, these studies have also highlighted their failure to reduce seroconversion to HSV-2 in high-risk populations. Nevertheless, his work has helped develop comprehensive methodologies for the clinical amd immunological assessment of newer vaccine formulations which have proved successful in animal models. The live attenuated Towne strain vaccine has been shown to prevent severe human cytomegalovirus (CMV) infection in transplant recipients. More recently, subunit antigens and virus vector vaccines against CMV and Epstein Barr virus (EBV) have been devised. Novel attempts to present viral antigens now include the development of plasmid expression vectors for viral nucleic acids and genes as well as engineered live vectors for viral antigens. These new technologies may allow future vaccines to be devised, not only against the 5 well characterised herpes viruses, but also against the recently recognised herpes viruses 6, 7 and 8 whose full clinical spectrum is still unknown.

The purpose of this study was to evaluate the immunogenicity of a herpes simplex virus type I (HSV-1) antigen preparation, obtained following zwitterionic detergent treatment of virus, and incorporation of the antigens into either immunostimulating complexes (ISCOMs) or non-ionic surfactant vesicles (NISV) delivery systems. Using Balb/c mice the ISCOM and NISV HSV-1 vaccines were assayed for their capacity to induce and enhance both the humoral and cellular immune responses, and to elicit protection against both homologous and heterologous virus challenge. The serum from animals vaccinated with either the NISV or the ISCOM HSV-1 antigen preparation, were found to contain high levels of total IgG and IgG1 and IgG2a subclass antibodies. In addition, both preparations were found to induce high neutralizing (NT) antibody levels following a two immunization protocol and to provide some protection against homologous and heterologous HSV challenge infection. Lymphoproliferative responses were observed in cultures of splenocytes from mice immunized with both HSV-1 NISV vaccine and HSV-1 ISCOMs vaccine, following various antigenic stimuli in vitro. In general, these were most marked in animals immunized with the HSV-1 NISV preparation, and particularly so when the splenocytes were stimulated in vitro with live HSV-1. Both the NISV and ISCOM HSV-1 vaccines were found to have induced interleukin 2, interleukin 10 and interferon-gamma in spleen cell culture supernatants, although again, the highest responses in general were observed in supernatant fluids from spleen cell cultures from animals immunized with the HSV-1 NISV preparation. These results suggest that a wide range of immune activity can be elicited by HSV-1 antigens presented to the immune system of mice in these formulations.

The vaccine potential of a genetically disabled Herpes Simplex Virus Type 1 virus (DISC HSV-1) was investigated in the guinea pig model of intravaginal (i.vag.) HSV-2 infection. Three mucosal vaccination routes, i.vag., intranasal (i.n.) and oral, were compared for their ability to protect guinea pigs from challenge with wild-type HSV-2. Each was effective, particularly the i.n. route, which almost completely abolished primary disease. This was accompanied by significantly lower challenge virus titres in vaginal swabs collected from the vaccinated animals. In all cases, vaccination with the inactivated virus preparation provided substantially less protection from disease than the live DISC HSV-1 by the equivalent route. Antibody levels in serum and vaginal washes were measured both after vaccination and challenge by ELISA and neutralization tests. The highest titres were observed following administration of the DISC HSV-1 vaccine by the i.n. route. Significant increases in IgA and IgG in vaginal wash fluids were also found in these vaccinated animals.

Immunostimulating complexes (ISCOMs) were prepared with mixtures of antigens from influenza A virus (A/PR/8/34 or A/Sichuan/2/87) and herpes simplex virus type 1 (HSV-1), and were characterised by enzyme linked immunosorbent assay (ELISA) and electron microscopy using double-labelling immunogold techniques employing monoclonal antibodies to influenza or HSV-1 glycoproteins. The immunogenicity of the mixed antigen ISCOMs was evaluated in mice, following administration by the subcutaneous route, by measuring the total and subclass IgG antibody responses. Protection of these animals against challenge with live influenza A/Sichuan virus or live HSV-1, was compared with that induced by immunization with aqueous mixed antigen preparations. It was found that relatively high humoral responses to both influenza and HSV antigens, and increased levels of protection to both influenza and HSV viruses were elicited in mice receiving the mixed antigen ISCOM preparation compared to those observed in animals receiving the mixed aqueous subunit preparation. The findings also indicate that antigens from more than one virus can be used in an ISCOM formulation to produce immunity and protection.

Experiments were carried out to examine the initial feasibility of immunizing infants and children with inactivated influenza virus vaccine combined with diphtheria-tetanus-pertussis (DTP) vaccine. Groups of mice were immunized with saline vaccine or vaccine mixed with DTP: three doses of vaccine were given 3 weeks apart, and the antibody response and resistance to challenge infection were tested 3 weeks after the 1st and 3rd immunizations. The results showed that the antibody response and immunity to challenge virus infection were significantly greater for mice given vaccine in DTP than for mice given saline vaccine alone. Comparison of the response to graded doses of vaccine in saline or DTP indicated that vaccine in DTP was > 250-fold more effective in inducing serum antibody and protection than saline vaccine alone. The enhancing activity of DTP was significant for the alum component alone; however, most of the adjuvant effect was from the antigen components of the DTP vaccine. The results suggest that immunization against influenza in infants and young children could be achieved by combining small amounts of influenza antigen with DTP vaccines; however, the present results have been obtained in mice, and, since the responses to vaccines and adjuvants vary from species to species, the present results cannot be used to indicate similar results in human volunteers. The results indicate the potential value of an immunization procedure which should be tested in volunteers and which could provide a simple strategy for the immunization of at-risk infants and children against influenza.

The immunogenicity and protective efficacy of an influenza A subunit vaccine preparation administered to mice in an aqueous form, or presented as immunostimulatory complexes (ISCOMs), liposomes or with Freund's complete adjuvant (FCA), were assessed in comparative studies with live infectious virus. Both intranasal and parenteral routes of administration were assessed. An enzyme-linked immunosorbent assay (ELISA) was used to measure nasal wash and serum antibody responses in groups of unprimed mice, while protection was determined by the recovery of homologous influenza virus from mouse nasal washes and lung homogenates following challenge infection by the intranasal route. The results showed that parenteral administration of the influenza antigen preparations induced variable levels of both local and systemic antibodies at weeks 3, 7 and 22 postimmunization. Although the overall greatest levels of antibody and protection were elicited in mice following live virus infection, formulation of influenza surface haemagglutinin (HA) and neuraminidase (NA) proteins into ISCOMs elicited high and persistent antibody responses and provided relatively good protection of the upper and lower respiratory tracts of these animals. The results also show a relatively poor effect of the subunit antigen preparations in promoting humoral immune responses and protection irrespective of the nature of their presentation, when given by the intranasal route.

Reconstituted influenza virus (A/PR/8 strain) envelopes (RIVE) and influenza virus (A/Sichuan/87 (H3N2) strain) surface antigens were entrapped in dehydration-rehydration vesicles (DRV liposomes) composed of egg phosphatidylcholine (PC) or distearoyl phosphatidylcholine (DSPC DRV) and equimolar (32 mumol) cholesterol. Entrapment values for RIVE were 31.2 (PC) and 29.4% (DSPC DRV) of the material used. Corresponding entrapment values for the A/Sichuan/87 strain antigens were 40.7 and 39.3%. Balb/c mice injected intramuscularly with PC or DSPC DRV liposomes containing 0.1 and 1.0 microgram RIVE exhibited primary (higher dose only) and secondary responses (IgG1) which were significantly higher than those obtained in mice injected with identical amounts of non-entrapped RIVE. Significantly higher secondary responses were also observed for the IgG2a and IgG2b subclasses. In experiments designed to assess the effectiveness of DRV liposomes as a carrier of influenza virus antigens in a potential vaccine, hamsters were immunized intramuscularly with 0.1, 0.5 and 5.0 micrograms of free or liposome-entrapped influenza A/Sichuan/87 surface antigens. Results showed increased haemagglutination inhibition (HI) antibody levels in terms of both primary (0.5 and 5.0 micrograms doses) and secondary (all doses) responses in the sera of animals treated with the liposomal formulations. DSPC compared with PC DRV exhibited greater adjuvanticity when the lower doses of antigens were used.

The capability of a herpes simplex virus (HSV)-1 ISCOM vaccine to protect against intravaginal HSV-2 challenge infection in guinea-pigs is described. The protective efficacy of the HSV-1 ISCOM vaccine is compared with that of a purified, aqueous HSV-1 antigen preparation administered using a similar immunization schedule. The results show that female guinea-pigs immunized with two doses of HSV-1 ISCOM vaccine, each consisting of 20 μg of protein given 2 weeks apart responded with high ELISA and neutralization antibody titres, and are almost completely protected against the clinical effects of intravaginal challenge with 10 5.2 TCID 50 of HSV-2. This cross-protection is significantly greater than that observed in guinea-pigs immunized with a single dose of HSV-1 ISCOM vaccine, two doses of aqueous HSV-1 antigen preparation or two doses of a mock ISCOM vaccine. However, none of the vaccine preparations completely prevented HSV-2 replication following challenge. Western blot and radioimmunoprecipitation of sera from immunized guinea-pigs show the HSV-1 ISCOM vaccine preparation to contain the major HSV-1 glycoproteins. These findings are discussed in relation to the value and potential use of HSV-1 ISCOM vaccine in humans.

The preparation and characterization of an immunostimulating complex (ISCOM) preparation containing several HSV-1 glycoproteins, including the major glycoproteins B and D is described. The multi-glycoprotein HSV-1 ISCOM preparation was obtained from a gradient-purified aqueous HSV-1 antigen preparation following extraction from infected cells using a zwitterionic detergent. With polyclonal and monoclonal antibodies to HSV-1 glycoproteins in enzyme-linked immunosorbent assay, SDS-polyacrylamide gel electrophoresis and radioimmunoprecipitation techniques, the HSV-1 ISCOM preparation was shown to contain glycoproteins B, C, D, E, H and I, although further, additional proteins were also present. The DNA content of HSV-1 ISCOMs was determined using a 3H-thymidine labelling method. The protein and DNA contents of the HSV-1 ISCOM preparation are discussed with reference to the potentialities of the preparation as a vaccine for use in human beings.

To determine their efficacy as barriers to the passage of lipid-enveloped viruses, wound dressings were exposed to known concentrations of Semliki Forest virus (SFV) at their inner surfaces for varying periods of time. The dressings were tested such that their outer surfaces were maintained in either a dry or a wet environment. Out of a total of 120 dressings each tested at four time points under wet conditions, virus was found to have penetrated on only one occasion. Similarly, virus penetration was noted in only a single test out of 442 carried out on 59 dressings under dry conditions. The dressings under test thus proved highly effective barriers to passage of a lipid enveloped virus.

HSV-1 antigen preparations solubilised from Vero cells by using either the non-ionic detergent Nonidet P40 or the zwitterionic detergent Empigen BB, and purified on sucrose density gradients or over a sucrose cushion, were tested by ELISA with anti-HSV-1 glycoprotein monoclonal antibodies and by radioimmunoprecipitation (RIP) with polyclonal HSV-1 antiserum. Amongst several proteins detected in these preparations, the four major HSV-1 glycoproteins, gB, gC, gD, and gE, were found to be present. Differences between NP40 or Empigen-solubilised HSV-1 antigen preparations with respect to two of these glycoproteins, gB and gE, were detected by using a small panel of monoclonal antibodies. Comparative studies in mice showed the Empigen-solubilised HSV-1 antigen preparations elicited greater antibody responses and greater protection against lethal HSV-1 challenge infection than the NP40-solubilised preparation.

Methods to obtain better results with hydraulic fracturing in low permeability sandstone formations are constantly being developed. Fracturing techniques employed are often cyclic in nature, catalyzed to a large extent by gas and oil prices and corresponding costs of the stimulation treatments. "Optimization" of the fracturing approach may often seem arbitrary and raise the question: "Optimum …. Compared to what?" Technology for improvements in hydraulic fracturing results is advancing at a rapid pace from the 3-D Seismic measurements used to define pay structure all the way to improved real-time mapping of fracture propagation. Extensive data gathering projects, however, are frequently big budget items and require dedication of manpower and resources. Therefore, such data measurement often tends to be reserved for projects of a regional scope. This paper discusses several case studies of fractured wells in the Cotton Valley formation in East Texas and describes systematic changes in fracturing techniques used to improve treatment success. Comparisons of production data (short term and long term) are used to illustrate the degree of improvement. Readily available information (open hole logs, RA Tracer Logs, and PLT logs) was used systematically in this study.

Nasal influenza vaccination may prove to be a good alternative to parenteral injection because of the enhancement of the mucosal immune response and the ease of vaccine administration. This study investigated the use of chitosan, a bioadhesive polymer, as a nasal delivery system with inactivated, subunit influenza vaccine. Subjects received nasally 15 or 7.5 microg of the standard inactivated trivalent influenza vaccine with chitosan or 15 microg of the same vaccine intramuscularly. Serum haemagglutination inhibition (HI) titres for all three vaccine components were measured prior to, and at time points up to 14 weeks after dosing. Serum HI titres following intranasal vaccination with the nasal chitosan-influenza vaccine met the criteria set by the Committee for Proprietary Medicinal Products in terms of seroprotection rate, seroconversion rate and mean fold increase of HI titre for at least one of the three antigens in the vaccination schedules used. These data show that nasal immunisation with chitosan plus trivalent inactivated influenza is a potentially effective, easily-administered form of vaccination.

Although shown to be a potent stimulator of serum antibody responses in animal models, the adjuvant immuno-stimulating complexes (ISCOMs) showed little adjuvant effect for inactivated influenza vaccines in a volunteer study. The result may be the non-comparability of the studies: animal studies were carried out chiefly in unprimed mice, while volunteers are mostly primed by previous infection and/or immunization. To test this, Balb/C mice were infected with influenza viruses or immunized with inactivated influenza vaccine, and subsequently given inactivated vaccine in saline or incorporated into ISCOMs. The serum in antibody responses was measured 1 month after immunization. The results confirm the adjuvant activity of ISCOM in unprimed mice, and show a marked reduction in adjuvant activity for primed mice. We argue that ISCOMs are important to prime the T cell response necessary for the serum antibody response to saline vaccine, but largely unnecessary where priming has been accomplished by prior exposure to influenza antigens. Further, the value of ISCOMs may lie in promoting antibody responses in unprimed subjects, and not in enhancing antibody titres.

Influenza is responsible for at least 3000-4000 deaths per year in the UK. During epidemics, death rates from pneumonia and influenza are much higher in elderly than younger people. With vaccination, the substantial medical and economic costs associated with influenza epidemics can be avoided. Countries with blanket vaccination have seen major reductions in morbidity and mortality. The vaccine is safe and effective in elderly people. Vaccination programmes for health-care workers who have contact with elderly people have also been shown to be effective in reducing deaths from influenza among this group.

Herpes simplex encephalitis (HSE) is a rare but very serious disorder caused by herpes simplex type 1 virus (HSV-1). Treatment with acyclovir decreases mortality but many patients still suffer cognitive impairment subsequently. A vaccine against HSV1 would therefore be of great value. HSV-1 has been implicated also in Alzheimer's disease (AD): we established that HSV1 resides in the brain of about two thirds of AD patients and aged normal people, and that in carriers of the type 4 allele of the apolipoprotein E gene, it is a strong risk factor for AD. Thus a vaccine against HSV-1 might prevent development of AD in some cases. To find whether a vaccine of mixed HSV-1 glycoproteins (ISCOMs), which protects mice from latent HSV-1 infection of sensory ganglia, prevents HSV1 latency in the CNS, ISCOM-vaccinated or unvaccinated animals were infected with HSV-1. Using polymerase chain reaction (PCR) we detected HSV-1 in brain from 16 of 39 unvaccinated mice (41%), but only 3 of 41 vaccinated mice (7%) (P < 0.001). Thus, ISCOMs protect the CNS also, suggesting their possible future usage in humans.

It is likely that recurrent infections with HSV-2 (or HSV-1) are influenced by local levels of immunity at mucosal surfaces, when virus reactivated from the latent state is infecting mucosal epithelial cells. Increasing the levels of cellular and humoral immunity through immunisation and maintaining such increased levels, may reduce establishment and spread of reactivated virus at the local site, thereby ameliorating recurrent disease symptoms. The use of HSV-2 antigens incorporated into immunostimulating complexes (ISCOMs) for immunisation of mice previously infected with HSV-2 was investigated in the present study. Prophylactic administration of HSV-2 ISCOM vaccine to mice elicits local antibody detectable in nasal washings, serum antibody and the presence of cytokines IL-2, IFN-gamma and IL-4 in supernatants from spleen cell cultures stimulated in vitro with HSV-2 antigens. Use of the same vaccine in mice infected previously with HSV-2, results in increased levels of total and subclass serum ELISA antibody and also increased levels of serum neutralising antibody. Treatment of HSV-2 infected mice with the HSV-2 ISCOM vaccine also induces higher levels of the cytokines IL-2, IFN-gamma and IL-4, in in vitro stimulated spleen cell cultures. Challenge with a lethal dose of HSV-1 showed that mice previously infected with HSV-2 and subsequently given two doses of HSV-2 ISCOMs vaccine were protected.

The immunogenicity of a type 2 herpes simplex virus (HSV-2) antigen preparation following its formulation into immunostimulating complexes (ISCOMs) or non-ionic surfactant vesicles (NISV) was investigated in a murine model. The immune responses induced by each formulation were characterised by antigen specific total and subclass serum responses, and by lymphocyte proliferation and cytokine (interleukin-2 (IL-2), interleukin-4 (IL-4) and interferon-gamma (IFN-gamma)) production by in vitro restimulated spleen cells. The degree of protection afforded to mice by these various HSV-2 vaccine preparations against homologous (HSV-2) and heterologous (HSV-1) challenge infection was also determined. The findings suggest that formulation of the HSV-2 glycoprotein antigens with ISCOM or NISV delivery vehicles, and the methods used to prepare these formulations, influenced the immunogenicity of the final preparation. Higher IgG2a and neutralising antibody levels, IL-2 and IFN-gamma levels and lymphoproliferative responses were noted in mice immunised with the HSV-2 ISCOM formulated vaccine preparation. Furthermore, although HSV-2 antigens formulated in dehydration-rehydration NISV, or entrapped in NISV by freeze-thawing at 30 degrees C (HSV-2 NISV 30), also elicited relatively high antibody, IL-2 and IFN-gamma levels and relatively high lymphoproliferative responses, formulation of HSV-2 antigens by freeze-thawing with NISV at 60 degrees C (HSV-2 NISV 60) did not. There were no differences between any of the HSV-2 vaccine formulations in terms of IL-4 induction in in vitro stimulated spleen cell cultures. Almost complete protection against HSV-2 challenge was afforded by the HSV-2 ISCOM preparation, while partial protection against challenge infection was afforded by the HSV-2 NISV 30 vaccine formulation. The findings are discussed in relation to the nature of the immune mechanisms, particularly Th1- or Th2-like responses, that may be elicited by HSV-2 antigen preparations formulated into various delivery systems and the relevance of these immune responses to protection against HSV infection in the murine model.

The development of improved vaccines against epidemic and pandemic influenza virus infection remains a priority in vaccine research. Killed vaccines given by injection are both cost-effective and induce immunity; however, their limitations are well known. Live vaccines have been in development for many years, but difficulties and safety concerns have prohibited their licensing in Western countries. However, the newer technologies of vaccine development, including DNA vaccines and attenuated virus vaccines produced by reverse genetics, remain a hope for the future. With these problems in mind, emphasis has been given to the development of inactivated vaccines that are administered intranasally, either as repeated doses of saline vaccine or in conjunction with suitable carriers or adjuvants. This review describes these latter developments and concludes that this approach offers advantages and should be vigorously researched.

The primary route of entry into the body for many viruses, including herpes simplex virus type 2 (HSV-2) and the influenza virus, is via mucosal surfaces. It follows that, depending on the numbers of virions deposited on such surfaces, their infectious quality and distribution over the mucosal surface, the existence or early mobilisation of specific defence mechanisms at the time of this contact, supplementing or enhancing any non-specific resistance mechanisms already operating at these sites may tip the balance in favour of the host and result in elimination of the invading virus before it can become established in the tissues and produce disease symptoms.

Following the demonstration of the strong adjuvant effect of whole-cell DTP vaccine (wDTP) on the immune responses to influenza subunit vaccine, studies were undertaken to identify the component of wDTP responsible for the adjuvant effect, and to determine if acellular DTP (aDTP) vaccine was as effective since it is less reactogenic and likely to replace wDTP for primary or secondary immunisation. In addition, wDTP and aDTP were directly compared in a dose-response study. Experiments in mice indicated that the adjuvant effect of wDTP resided in the LPS component of B. pertussis , since purified LPS enhanced the IgG antibody response, the IgG subclass response and protection to the same level as wDTP. An adjuvant effect was detected using aDTP, but was statistically less pronounced than wDTP by a factor of some 100-fold. These results suggest that immunisation against influenza in infants and young children can be achieved by combining small amounts of influenza antigen with wDTP or LPS, and to a lesser extent by combining vaccine with aDTP. However, these results were obtained in mice and should be confirmed in man since species vary considerably in response to adjuvant.

The vaccine potential of a mutant herpes simplex virus (HSV) type 1, with a deletion in the glycoprotein H (gH) gene, was evaluated. The virus requires a gH-expressing cell line for multi-cycle growth but can complete a single cycle of infection in noncomplementing cells. Such viruses, termed DISC (disabled infectious single cycle) viruses, should be safe, yet still able to stimulate humoral and cell-mediated responses against a broad range of virus antigens in vaccinated hosts. Prophylactic vaccination of guinea pigs with DISC HSV-1, by ear scarification or direct infection of the vaginal mucosa, afforded a high degree of protection against HSV-2-induced primary genital disease and reduced significantly the frequency of subsequent disease recurrence. There was also a trend toward reduced recurrence following therapeutic vaccination of animals already infected with HSV-2. DISC HSV vaccination, therefore, offers an effective route for control of HSV disease.

Unprimed mice and mice primed by prior infection with an H1N1 subtype of influenza virus were used to assess the total and subclass IgG serum antibody responses to influenza virus A/Sichuan/2/87 (H3N2) surface haemagglutinin and neuraminidase proteins incorporated into four different formulations of liposomes. Only one of these liposome preparations, DSPC(B), induced greater total IgG, and subclass IgG1 and IgG2a antibody levels, in sera from both primed and unprimed mice than the aqueous A/Sichuan surface preparations alone administered at equivalent levels of haemagglutinin protein. The same DSPC(B) liposome formulation of A/Sichuan antigens was also the only preparation found to elicit levels of IgG2b and IgG3 subclass antibodies above baseline values in these animals.

Five spermicides including nonoxynol-9 were assessed under in-vitro conditions, for their inhibitory activity against two viruses capable of spread by sexual intercourse, herpes simplex virus type-2 (HSV-2) and the human immunodeficiency virus type-1. A further eight commercially-available spermicidal preparations containing varying concentrations of either nonoxynol-9 or nonoxynol-11 were also assessed for activity against HSV-2. All spermicides and spermicidal preparations tested showed inhibitory activity against both viruses over periods of time ranging from 30 sec to 5 min. This activity was dependent on the concentration of spermicide to which the viruses were exposed.In England, microbiologists at the University of Sheffield Medical School diluted herpes simplex virus-2 (HSV-2) in Eagle's Basal Medium + 5% fetal calf serum to a final concentration of 1 x 100,000 plaque-forming units/ml and HIV-1 in RPMI 1640 medium to a final concentration of 1 x 100,000 syncytium-forming units/ml. They wanted to study the inhibitory activity of the spermicidal agents nonoxynol-9, nonoxynol-11, benzalkonium chloride, menefegol, and sodium docusate. A concentration of pure nonoxynol-9 of .025% was the minimum concentration needed to inactivate HSV-2 in vitro, which was 4-160 time slower than concentrations of proprietary nonoxynol-9 preparations (2-8%) needed to have the same effect in vitro. The minimum concentration of nonoxynol-11 and benzalkonium chloride needed to inactivate HSV-2 in vitro was also .025%. Sodium docusate had a 2.5 times greater ability to inactivate HSV-2 in vitro than did nonoxynol-9 and nonoxynol-11 and benzalkonium chloride (minimum concentration = .01%). The minimum concentration of menfegol needed to inactivate HSV-2 in vitro was lower than that of the other spermicidal agents (.1%, a concentration 4 times lower than that of nonoxynol-9). All the spermicidal agents effective at inactivating HSV-2 did so in at least 30 seconds. The minimum concentration of nonoxynol-9 needed to maximally inactivate HIV-1 in vitro was .025%. it did so in 30 seconds. The lowest concentration of sodium docusate, menfegol, and benzalkonium chloride necessary for in vitro HIV-1 inactivation was also .025% (minimum time: 30 seconds for the first 2 spermicides and 2.5 minutes for the third). These findings suggest that spermicidal agents have virucidal capabilities.

Comparative studies on the local IgA, and circulating IgG subclass antibody responses of mice to A/Sichuan/2/87 (H3N2) influenza virus surface antigens administered with different carrier or delivery systems by the parenteral route, were carried out. The results obtained were compared with the responses observed following live influenza virus infection, and the protection afforded to these animals by these various preparations determined. Infection with live virus elicited early and high levels of protection against homologous virus challenge and this correlated with both local IgA and circulating IgG2a antibody levels. When incorporated into immunostimulating complexes (ISCOMS), A/Sichuan surface antigens promoted high levels of local IgA and circulating IgG1 antibody, and achieved a more rapid and more solid immunity against homologous virus challenge infection, than that elicited by the same surface antigens administered alone or together with Freund's complete adjuvant or alhydrogel.

The effect of immunisation with an HSV-1 antigen preparation (containing at least 6 viral glycoproteins) on primary infection with HSV and the establishment of latency, was assessed in two mouse models (involving either skin or corneal challenge with virus). The vaccine preparation, given either with Freund's complete adjuvant or aluminium hydroxide gel or in the form of immunostimulating complexes (ISCOMS), induced high ELISA antibody responses (highest with HSV as the ISCOM preparation) and low levels of neutralising antibody. In both models, immunisation with the HSV ISCOM preparation significantly reduced the incidence of zosteriform spread of virus and the severity of disease and, in some cases, the incidence of latent infection in sensory ganglia. In the eye model it was possible to show that immunisation with the HSV ISCOMS restricted the establishment of latency almost entirely to the ophthalmic part of the trigeminal ganglion. Protection from establishment of latency correlated with prechallenge antibody levels.

A HSV-1 antigen preparation obtained by zwitterionic detergent-extraction of HSV-1 infected Vero cells was investigated for its ability to protect mice against establishment of latent infection in the trigeminal ganglion following HSV-1 challenge by ear scarification. The antigen preparation was shown to induce antibody production and correlation between pre-challenge antibody level and establishment of latency was found. Although immunization did not reduce the overall incidence of ear erythema following challenge the duration of erythema was significantly decreased. Latency in the trigeminal ganglion was also significantly reduced in vaccinated mice and in addition, a correlation was found between the duration of erythema and latency, there being significantly more latent infections in mice with erythema of three or more days duration, than in those with erythema lasting less than three days.

The behaviour of three recombinant strains of influenza virus A/England/939/69 and A/PR/8, together with the parent A/England/939/69 strain, all of known virulence to man, were examined in ferrets. For the virulent A/England/939/69 strain, the peak temperature response and the time of peak virus excretion occurred 48 hours post-infection, and peak protein and secretory antibody responses occurred on days 7 and 9. In contrast, the peak temperatures and maximum virus excretions for the recombinant attenuated viruses, clones 7 and 6a, occurred on day 3 and 6, and the maximum protein and nasal antibody responses on days 9 and 11, respectively. Although the peak responses for these strains were distinct, the magnitudine of the symptoms was comparable. Similar studies with the over attenuated clone 64C virus showed a milder infection. In addition, the maximum temperature response and virus excretion occurred on day 5 post-infection, and the maximum nasal protein concentration was found on day 11 after virus infection. These differences between the strains were only apparent when animals were given an inoculum of 10 EID50; no differences were observed between the four strains when larger inocula were used. The results suggest that virus infection of ferrets was distinct for strains of differing human virulence, and that the ferret model may provide a test for virulence for man.

SUMMARYGroups of volunteers were immunized with one of three influenza virus vaccines, and the resistance to challenge infection with attenuated influenza A (H1N1) virus was measured 8 months later. The vaccines were aqueous subunit influenza A/USSR/77 (H1N1) vaccine, aqueous subunit influenza B/Hong Kong/73 vaccine, or attenuated influenza virus A (H1N1) vaccine. The B virus vaccine was included as a control to assess the incidence of natural A virus infection during the study period. A proportion of the B virus vaccinees had pre-existing A (H1N1) virus antibody and were used to study the immunity conferred by natural infection to the live virus challenge. The serum antibody responses were measured at 1 and 8 months after immunization. The results showed that all the vaccines induced serum HI antibody in a proportion of the volunteers; however, after 1 month, higher titres of serum antibody were found in volunteers given inactivated A vaccine than in those given live attenuated A virus vaccine. Eight months post-immunization the titres of serum antibody in volunteers given inactivated vaccine had declined significantly, but there were no changes in the antibody titres of those given live virus vaccine. The incidence of infection by the challenge virus at 8 months post-immunization was directly related to the serum antibody titres 1 month post-immunization; no evidence was obtained to suggest that those given live virus vaccine had a more solid immunity than those given inactivated vaccine.

Two separate, double-blind studies were carried out in volunteers to compare the reactogenicity of, and serum antibody responses to, whole or ether-Tween-split inactivated influenza virus vaccines. In both studies the ether-Tween-split vaccines induced a lower rate of reactions. The serum hemagglutination-inhibiting (HAI) antibody response of volunteers to the A/Scotland/74 component of the split vaccine used in the first study was significantly greater than that following inoculation of A/ Scotland/74 whole-virus vaccine. The neuraminidase-inhibiting (NI) antibody responses of the volunteers to each vaccine were similar. In the second study, a markedly better NI antibody response to the influenza A virus component was seen following immunization with split-virus vaccine, but the HAI antibody response to both split and whole vaccines was the same. In both studies the serum HAI antibody responses to the B/Hong Kong/73 component of the vaccines were similar. Challenge of the volunteers with attenuated influenza viruses homologous to the influenza A component of the vaccines showed both types of vaccines to be protective.

Guinea-pigs immunized with a zwitterionic detergent-extracted HSV-1 antigen preparation responded with EIA and NT serum antibody titres that were significantly greater than those elicited by a non-ionic detergent-extracted antigen preparation inoculated using a similar dosage schedule. Following intravaginal challenge of the guinea-pigs with HSV-2 (strain SH/B), there was no statistically significant difference in the protection afforded to these animals by the two antigen preparations, although the results indicated the zwitterionic detergent-extracted HSV-1 antigen preparation to be slightly superior in this respect. In mice, the zwitterionic detergent-extracted HSV-1 antigen preparation elicited an EIA antibody response and partial protection against homologous virus challenge. The relevance of these animal models to determination of immunogenicity and efficacy of HSV vaccines prepared for use in man, and the reasons for the differences in immunogenicity of these HSV-1 antigen preparations in guinea-pigs, are discussed.

The ability of inactivated influenza A vaccines to induce serum HI antibody and immunity to challenge infection was studied in hamsters and in volunteers. Groups of hamsters were immunized with 200 IU of influenza virus A/Scotland/74, A/Port Chalmers/73, A/England/72, or A/Hong Kong/68. The serum HI antibody response of animals to, and immunity to challenge infection was directly related to the known relationship between the vaccine and test viruses. Thus, hamsters given A/Hong Kong/68 or A/England/72 vaccine produced serum HI antibody and immunity to A/Hong Kong virus infection, and animals given A/Scotland/74, A/Port Chalmers/73, and A/England/72 produced antibody and immunity to A/Scotland infection.

A comparison between the enzyme-linked immunosorbent assay (ELISA), the single radial haemolysis (SRH) and the complement-fixation (CF) tests for the measurement of mumps virus antibodies in a sero-epidemiological survey is described. The ELISA, although showing a degree of correlation with the SRH and CFT, was found to be a more sensitive and also a more appropriate test compared to the SRH and CFT for this purpose, as the mumps virus antibody incidence and levels determined using ELISA, correlate most nearly with those antibodies measured using a neutralisation (Hd-NT) test. Using the ELISA for determining antibodies to mumps virus in sera representative of all age groups of the populations of Baghdad, Iraq, and Sheffield, England, it was found that the incidence of such antibodies was significantly higher in sera from adults in Baghdad, while in younger individuals such a difference was not apparent. Further differences in the levels of mumps virus antibody, as measured by ELISA, were observed in sera from various age groups of the Baghdad and Sheffield populations. Possible reasons for these differences, and the relative values of the tests used for measuring mumps virus antibody in the present study are considered in the discussion.

The growth characteristics of a series of influenza A viruses in the turbinates and lungs of hamsters was measured: in addition, the susceptibility of hamsters to infection by these viruses was also determined. These two criteria were used to give estimates of the growth potential of influenza viruses in hamsters, and the results were related to the incidence of transmission of virus from inoculated hamsters to cage-contacts. The results showed that strains of influenza virus reported as virulent for man tended to grow to higher titres in hamster nasal washings and lungs; were more infective for hamsters when inoculated by the intranasal route; and showed a high incidence of spread to cage-contacts. The methods could provide valuable measurements of virus attenuation and transmissibility for man, and the further exploitation of these techniques could facilitate the production and licensing of live, attenuated influenza virus vaccines.

Hamsters previously infected by influenza viruses, have been shown to have a cell-mediated immune response, as measured by the macrophage migration inhibition test. The participation of spleen cells in the protection of recipients against homologous influenza virus infection was also demonstrated using adoptive transfer experiments. However, the protection achieved by spleen cell transfer was marginal and not observed in every animal. The time at which the spleen cells were transferred following infection, and their number, affected the outcome. Evidence suggesting that transferred spleen cells protected recipient hamsters through specific antibody is presented.

Several wild-type influenza A strains together with recombinants derived from these strains, were tested for sensitivity to amantadine using the in vitro techniques of inhibition in egg-bit culture and plaque reduction in MDCK cells. The results obtained were analysed with reference to the derivation of the recombinants. Susceptibility to amantadine was related to the gene coding for matrix protein, and these data are in agreement with previous reports of studies using other series of influenza viruses.

Guinea pigs were inoculated by intranasal inoculation with unadapted, influenza virus A/England/42/72, and virus was recovered from nasal washings between 3 and 10 days post-inoculation. Infected animals did not exhibit a febrile response to infection, did not produce local antibody and produced only relatively low levels of serum antibody. However, they developed delayed-type hypersensitivity to influenza virus, demonstrable by both skin tests and macrophage migration inhibition tests, which was similar to that of man. The relevance of the influenza virus specific delayed hypersensitivity in immunity to infection was examined in this model. Guinea pigs previously infected with virus or passively immunized with hyperimmune serum were relatively resistant to reinfection with influenza virus A/England/42/72. Inoculation of guinea pigs with spleen cells from immune donor animals, together with or without immune serum, did not give or enhance resistance to challenge virus infection. The results do not suggest a role for delayed hypersensitivity response in immunity to influenza virus infection.

Summary A group of 23 student volunteers were each inoculated intranasally with 400 IU of inactivated, freeze-dried A/England/42/72 vaccine. Only one volunteer showed a four-fold rise in serum HI antibody following immunization, and the mean increase in serum HI antibody (gmt) for all volunteers did not increase two-fold. Thirteen of the volunteers developed detectable levels of nasal wash neutralizing antibody after immunization; local antibody was most commonly found in volunteers who also produced a detectable but less than four-fold rise in serum antibody titre, and who produced nasal washings with relatively high concentrations of protein and secretory IgA. Four weeks after immunization, the vaccinees and a matched group of control subjects were inoculated with attenuated A/England/42/72 (MRC-7) virus. Evidence of infection was found in 14 of 23 (61 per cent) of control subjects and in seven of 23 (30 per cent) of immunized volunteers. This result showed a significant protection (P=0.04) against challenge virus infection for volunteers given intranasal vaccine.

A preparation was obtained from herpes simplex virus type 1 (HSV-1)-infected cells using a zwitterionic detergent, Empigen BB. The preparation was partially-purified either by ultracentrifugation over a cusion of 20% sucrose or on a sucrose density gradient. Partial characterisation of these materials by ELISA, using both polyclonal and monoclonal antibodies showed them to contain at least four major HSV glycoproteins, gB, gC, gD and gE. Comparison of Empigen-extracted HSV-1 antigen preparations with preparations obtained using the non-ionic detergents Nonidet P40 or Triton-X-100 indicate that, using conventional procedures, separation of glycoproteins, B, C, D, and E from unwanted proteins may be facilitated using the former detergent. Immunization of mice with Empigen-extracted, partially-purified or gradient-purified antigen preparations elicited good levels of antibody detectable by ELISA and a high degree of protection against both HSV-1 and HSV-2 challenge infection. Such protection could be achieved using aqueous antigen preparations, but was augmented using aluminium hydroxide gel as an adjuvant. In general, Empigen-extracted HSV-1 antigen preparations elicited higher ELISA antibody levels and more complete protection against HSV challenge infection than NP40 or Triton-X-100-extracted preparations. The value and usefulness of the detergent Empigen for obtaining HSV surface antigen preparations and the role of these as potential vaccines against HSV infections, is discussed.

The immunogenicity of zwitterionic detergent-disrupted influenza virus vaccine preparations, intact virus vaccine and vaccine preparations obtained by treatment of the intact virus with Triton-X 100 or cetyl trimethyl ammonium bromide (CTAB) was studied in hamsters and mice. In all experiments the intact, inactivated virus vaccine induced greater serum haemagglutination-inhibiting (HI) and neuraminidase-inhibiting (NI) antibody titres than the detergent-disrupted preparations. The serum HI antibody responses induced in hamsters and mice by Empigen-, Triton-, or CTAB-disrupted vaccines were similar, although more highly purified Empigen-disrupted preparations elicited marginally lower immune responses. In both animal species, all vaccine preparations afforded a similar degree of protection against homologous virus challenge.