Survey of Recombinant Viral Vaccines Currently under Development 6211 Herpes viruses

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Genital herpes is caused by the herpes simplex virus type 1 or 2 (HSV-1, HSV-2), or human herpes virus 1 and 2 (HHV-1, HHV-2). Subunit HSV vaccines are based upon two envelope glycopro-teins, gB and gD which have been shown to be strongly immunogenic and protective in animal studies (Stanberry 1991). Four separate formulations have been evaluated to date, all derived from CHO expression systems. Three vaccines developed by Chiron contained truncated HSV-2 gD absorbed to alum, gD with a muramyl tripeptide adjuvant, and a bivalent vaccine composed of gD and gB with MF59 adjuvant. Although all these vaccines were immunogenic, the first was only modestly protective, the second caused unacceptable side-effects while the third failed to protect (Corey et al. 1999; Langenberg et al. 1995; Straus et al. 1994; Straus et al. 1997). The fourth vaccine (from GlaxoSmithKline) is also based on truncated HSV-2 gD and alum combined with the novel adjuvant 3-de-O-acylated monophosphoryl lipid A. This preparation was well tolerated and induced potent humoral and CMI responses in Phase I/II trials (Leroux-Roels et al. 1994). A Phase III trial has indicated that although effective at prevention in women who are seronegative for both HSV-1 and -2 (but not those who are seroposition for HSV-1), it has no efficacy in men, regardless of their HSV serologic status. This may be due to the enhanced Th1 response noted in women (Stanberry et al. 2002). Further work is in progress to determine this.

HHV-8 is the most recently isolated human herpes virus, and is closely associated with Kaposi's sarcoma (Whitby & Boshoff 1998). The HHV-8 genome has many similarities to that of Epstein-Barr virus, and, should a vaccine be developed, this similarity may aid its development. Varicella Zoster

Varicella Zoster (VZV or human herpes virus 3 (HHV-3)) causes chickenpox. In adults, complications can develop leading to 20-25 deaths per year in England and Wales (Rawson et al. 2001). VZV is currently controlled by a live attenuated vaccine. Animal studies with recombinant VZV glyco-proteins B, C, E and I (gB, gC, gE and gI), have confirmed the role of antibody in protection, while the intermediate early protein IE62 appears to be implicated in the generation of VSV-specific CTL responses. A recombinant HSV-1 vector expressing either gE or IE62 induced antibody and CTL responses in mice, supporting the feasibility of a combined HSV/VZV vaccine (Lowry et al. 1997). Epstein-Barr virus

An effective vaccine against Epstein-Barr virus (EBV, or human herpes virus 4 (HHV-4)) would not only limit the outgrowth of latently-infected B cells in healthy individuals, but would also block the development of EBV-related cancers such as Burkitt's lymphoma, nasopharyngeal carcinoma and Hodgkin's disease. The virus membrane antigens, gp350 (and its derivative, gp220) and gp85, are the primary candidates for a vaccine. To date, nearly all candidate vaccines have been based on the B95-8 A-type strain gp350, which shares a high degree of structural and functional homology with B-type strains, suggesting that a B95-8-derived vaccine should be equally effective against B-type isolates (Lees et al. 1993). Gp350 has been shown to be the principal target of neutralizing antibody responses (Thorley-Lawson & Geilinger 1980), and recombinant gp350, either as a CHO-derived purified subunit or expressed from recombinant vaccinia vectors, has been shown to protect non-human primates (cotton-top tamarins) against EBV-induced B cell lymphomas (Morgan 1992). A recombinant vaccinia expressing latent membrane protein-1 from the BNLF-1 ORF was tested in a small group of human infants. All unvaccinated controls became naturally infected, but only 3/9 vaccinees became infected within 16 months (Gu et al. 1995). More recent studies have focused on the use of synthetic polytope vaccines, based on a concatomer of EBV-specific CTL epitopes from gp350 and gp85 (Khanna et al. 1999; Macsween & Crawford 2003) and Phase I/II trials are planned. Assuming that such trials are successful, it is envisaged that further trials will assess the effects of such vaccines on EBV-associated cancers. Here, the timescales are much longer, at least 10 years to show an effect against Burkitt's lymphoma and at least 50 years for nasopharyngeal carcinoma (Arrand 1998). Human cytomegalovirus

Human cytomegalovirus (CMV or human herpes virus 5 (HHV-5)) infection persists for life with periodic asymptomatic excretion of virus in body fluids. Up to 100 % of the population of non-industrialized countries are seropositive, while in industrialized countries the figure rises with age to approach 50 % in adulthood. CMV can complicate organ transplants, and is associated with retinitis in about 25 % of advanced HIV cases. However, the key justification for a vaccine is to prevent congenital CMV disease in newborns. Whole live virus vaccines based on the Towne strain tend to become heavily overattenuated and poorly immunogenic (Adler 1995), and alternative strategies using subunit and poxvirus vector vaccines are now under development. The CMV envelope glycoproteins gB and gH contain the majority of CMV epitopes against which neutralizing antibodies are induced. gB contains two major antigenic domains, AD-1 and AD-2, the latter being conserved among 12 clinical strains and contains epitopes for both CTLs and TH cells (Mitchell et al. 2002).

A recombinant gB antigen produced in CHO cells has been evaluated in Phase I trials in both adults and children (Frey et al. 1999; Mitchell et al. 2002; Pass et al. 1999; Wang et al. 1996). In adults, 100 % of vaccinees became seropositive and 98 % produced neutralizing antibodies after three immunizations of gB plus the adjuvant MF59. In children, three doses were also required for maximal effect, but antibody titres were six-times higher than those observed in adults. The vaccine was well tolerated in both groups. This is a most encouraging result, given that CMV transmission starts in infants. However, data from transplant patients suggests that CMI responses are critical in the control of CMV infection in this group. Analysis of T-cell responses in healthy CMV seropositive individuals has identified the tegument protein pp65 and the intermediate early protein IE1 as the immunodominant antigens for both CD4+ and CD8+ T-cells (Davignon et al. 1995; Wills et al. 1996).

Recombinant ALVAC canarypox vectors expressing gB and pp65 have been tested in Phase I trials for their ability to induce both neutralizing antibodies (Nabs) and CTLs. Three doses of ALVAC-CMV-gB induced only low levels of Nabs, but two doses of the viral vector, followed by one of attenuated Towne virus was much more effective (Adler et al. 1999). Two doses of ALVAC-CMV-pp65 induced long-lasting CTL responses in all vaccine recipients (Berencsi et al. 2001). Finally, a CHO-derived chimaeric IE1-pp65 protein has been shown to stimulate both CD4+ and

CD8+ T-cells from CMV-seropositive individuals in vivo (Vaz-Santiago et al. 2001). Phase II and III trials with all these vaccines are planned. Oncogenic viruses

On a global basis, cervical carcinoma is the second commonest female cancer (after breast cancer). There is a strong body of evidence to support the link between the presence of high-risk human papillomavirus (HPV) strains such as HPV-16 and -18 and cervical cancer (Phillips & Vousden 1998) and an effective anti-HPV vaccine would have an enormous impact worldwide.

In 2006, the FDA approved the first preventative HPV vaccine, Gardasil (Merck & Co.), while the similar GlaxoSmithKline HPV vaccine, Cervarix, is expected to be licensed in 2007. Both of these vaccines are recombinant virus-like particles (VLPs) composed of the major capsid protein, L1. L1 contains the major immunodominant neutralisation epitopes of the virus and induces high levels of protective neutralising antibodies. These protect girls and women against the two commonest HPV strains (HPV-16 and -18) implicated in cervical cancer, while Gardasil also targets HPV-6 and -11 which cause most cases of genital warts (Lowry & Schiller, 2006).

The products of the E6 and E7 open reading frames (ORFs) of HPV-16 have been implicated in the transformation of cervical epithelia into carcinoma, suggesting that immune responses raised against the E6/7 proteins may form the basis of a therapeutic vaccine to attack established tumours. The French biotechnology company, Transgene, has just completed Phase II trials of TG4001, a vaccine based on the E6 and E7 proteins, plus the cytokine IL2 delivered in an MVA vector (see: pipeline/iframe mva hpv il2.htm).

The human T-lymphotropic virus type 1 (HTLV-1) is estimated to infect between 10-20 million people worldwide and causes at least two types of disease - an aggressive T-cell malignancy, adult T-cell leukaemia/lymphoma (ATL) and a variety of chronic inflammatory syndromes, most notably HTLV-1-associated myelopathy. Where HTLV-1 is endemic (in the tropics and subtropics and among certain immigrant groups and intravenous drug users in Europe and North America), these are important causes of mortality and morbidity (Bangham 2000). Protection against HTLV-1 can be conferred to infants through maternal antibodies transferred by breastmilk. Thereafter, infection rates rise with declining levels of maternal antibody (Takahashi et al. 1991). Passive transfer experiments in rabbits have confirmed the role of antibody in protection (Sawada et al. 1991). The protective epitopes appear to be located on the env glycoprotein, gp46, and rodents can be protected against HTLV-1 infection by immunization with vaccinia-env, adenovirus-env or avipox-env (Bomford et al. 1996; Shida et al. 1987). CHO-derived HTLV-1 proteins protected pigtailed macaques against challenge with simian T-lymphotropic virus (Dezzutti et al. 1990) while cynomolgous macaques can be protected against HTLV-1 challenge by immunization with a recombinant vaccinia-gp46 vaccine which was found to induce both humoral and env-specific CTL activity (Ibuki et al. 1997).

Current opinion is that HTLV-1 infection is best controlled in endemic areas by transmission-blocking measures such as screening blood donors and refraining from breast-feeding, but where this is not possible, an env-based vaccine may be of some use. However, at the present time, no such vaccine is undergoing clinical trials.

More than 300 million individuals are carriers of hepatitis B virus (HBV) as identified by expression of surface antigen (HBsAg), resulting in 1 million deaths from the consequences of chronic HBV infection. Individuals who recover from acute HBV infection produce antibodies to HBsAg that confer lifelong protection. The current vaccine contains recombinant HBsAg synthesized in yeast or CHO cells and is safe and effective for all but a subgroup of immuno-compromised patients (the elderly, and infants of infectious mothers). HBV-infected hepatocytes secrete HBsAg as non-infectious, subviral particles in the form of 22nm spheres and tubules, and very similar structures are secreted by CHO cell lines stably transfected with the HBsAg gene. Attempts have been made to extend the effectiveness of the vaccine to the non-responding subgroup. Experiments in mice have indicated that immune responses to the major surface protein and pre-S domain are separate, and epitopes in the latter domain may enhance the response to the major surface protein (Milich 1988). Trials with pre-S2 containing vaccines have not overcome non-responsiveness, but more success (particularly with the elderly) has been noted when the pre-S1 domain is included (Neurath et al. 1986; Tron et al. 1989; Shouval et al. 1994).

Hepatitis C virus (HCV) is thought to be the major causative agent of non-A, non-B hepatitis, which can lead to cirrhosis, liver failure and liver cancer (Flint & McKeating 2000). Currently 170 million people are infected worldwide, and the only available treatment (interferon-a with ribavirin) is expensive and only moderately effective. A vaccine is thus a high priority, but efforts are hampered by the lack of a small animal model and the inability to routinely support HCV replication in vitro in cultured cells. Conventional neutralizing assays cannot, therefore, be performed, although there are conflicting reports that vesicular stomatitis virus (VSV)/HCV pseudotyped virus expressing combinations of E1 and E2 glycoproteins can be produced by transfected BHK cells (and possibly primary hepatocyte and human hepatoma cell lines) and be neutralized by homologous antisera (Buonocore et al. 2002; Lagging et al. 1998; Matsuura et al. 2001).

Chronic HCV carriers make antibodies against a variety of viral proteins, particularly the structural nucleocapsid proteins and the surface glycoproteins E1 and E2 (Flint & McKeating 2000; Lesniewski et al. 1993) and these antigens are currently the focus of a variety of potential vaccines. An early study showed that surface glycoproteins of HCV purified from recombinant vaccinia-transduced human cells were able to protect chimpanzees from a low challenge dose with the homologous strain (Choo et al. 1994). Further chimpanzee studies detected low levels of E2 antibodies, which did not protect the animals from heterologous HCV challenge but did protect them from subsequent chronic infection (Abrignani & Rosa 1998). An important study by Heile et al. (2000) examined the optimal form of the E2 antigen from the perspective of its ability to bind to the putative HCV receptor, CD81, and its capacity to generate antibodies that would inhibit the interaction of E2 with CD81. Soluble E2 truncated at amino acid 661 (E2-661) expressed in CHO cells only met the desired criteria when purified from the core-glycosylated intracellular fraction. The complex-glycosylated secreted fraction does not bind CD81 and does not elicit CD81/E2-blocking antibodies. Only glycosylated, monomeric non-aggregated E2 bound CD81, and protein immunization was more immunogenic than E2-encoding DNA vaccination. Identical findings were reported by Flint et al. (2000). Replication-deficient recombinant adenoviruses (Ad) efficiently expressed the core, E1 and E2 proteins in 293 cells, and induced high-titre, specific antibodies in mice (Makimura et al. 1996). A partially purified recombinant E1E2 fusion protein isolated from Ad/HCV-transfected HeLa cells raised antibodies to the E2, but not E1 protein in mice, while administration of a similar replication-deficient adenovirus expressing core, -E1 and -E2 proteins induced E2-specific CTLs, but not antibodies to E1 or E2. An adenovirus prime/protein boost regime induced both humoral and CMI responses to the E2 protein (Seong et al. 1998; 2001). The most interesting system currently under development involves the use of a replication-defective herpes simplex virus type-1 recombinant in which the HSV gH-encoding gene has been replaced by that of HCV E2-661. Vero, CR1 or Hep2 cells infected with the recombinant virus express high levels of correctly folded, non-aggregated (both intracellular and secreted) E2-661 protein which is highly reactive with sera from HCV-infected patients. Mice immunized with the HSV/HCV virus produced high titres of E2 antibodies. By contrast, most of the E2-661 protein produced by transient expression in 293 cells was found to consist of misfolded aggregates (Flint et al. 2000). The HSV/HCV system shows considerable promise as a potential vaccine. Haemorrhagic fever viruses

Viral haemorrhagic fevers (VHFs) represent a serious public health problem with recurrent outbreaks worldwide. The most widely distributed virus is dengue, causing 20 million infections per annum, and, despite the availability of an efficient live, attenuated vaccine, the spectre of yellow fever continues to haunt Africa and South America. In many cases, VHF control is primarily by elimination of the mosquito vector, but where the natural host is not known, as in the case of Ebola and Marburg virus, this is not feasible. Other important agents of VHF include Lassa virus and Japanese encephalitis virus (JEV), while a number of other agents (St Louis encephalitis virus, tick-borne encephalitis virus, Rift Valley fever virus, West Nile virus (WNV)) cause periodic localized epidemics. Unfortunately, with the exception of dengue, yellow fever and JEV, these diseases are a low commercial priority for vaccine manufacturers and comparatively little is known about their biology and immunopathology (Baize et al. 2001). However, the recent spread of WNV in the United States (3887 cases with 120 deaths by November 2006) has re-focused attention on this group of viruses.

Although tetravalent (DEN 1-4) live attenuated vaccines against dengue have been trialled in adults and children (Kanesa-Thasan et al. 2001), alternative control strategies have been slow to emerge. Monkeys have been protected against dengue-2 challenge by immunization with a vaccinia recombinant vector expressing truncated dengue-2 envelope (E) protein (Men et al. 2000), but protection against challenge could not be demonstrated in mice vaccinated with recombinant E-protein despite the induction of specific neutralizing antibody (Kelly et al. 2000). A more fruitful area of research concerns the use of chimaeric live-attenuated dengue vaccines, assembled using reverse genetics (Halstead & Deen 2002). Following the successful Phase I trial of a chimaeric yellow fever/Japanese encephalitis live attenuated vaccine (Monath et al. 2002), a number of similar vaccines have been made by inserting combinations of dengue virus 1-4 premembrane and E genes into the backbone of either the yellow fever virus 17D or attenuated DEN 1-4 (Guirakhoo et al. 2001). After promising results in non-human primates, Phase I trials are under way (summarized by Halstead & Deen 2002).

Approximately 100 million doses of yellow fever 17D live attenuated virus are produced annually, and are generally effective and well tolerated. However, incidences of extreme adverse reactions (including death) are not unknown, emphasizing the need for the mechanism of attenuation to be better understood (Marianneau et al. 2001).

Arenaviruses, such as Lassa fever, establish chronic infections in rodents leading to zoonotic transmission to humans. Of the VHFs, Lassa fever affects by far the largest number, being endemic in West Africa from Guinea to Nigeria. Although clinically severe, with up to 30 % mortality, infection confers life-long immunity, leading to the belief that a vaccine can be developed (FisherHoch & McCormick 2001). Viral clearance appears to be by cell-mediated mechanisms, and the presence of antibody to viral antigens is negatively correlated with survival (ter Meulen et al. 2000). The first genetically engineered vaccine candidates appeared in the early 1980s, based on recombinant vaccinia virus expressing the nucleocapsid (NC) or glycoprotein (GP) genes of Lassa virus. Although guinea pigs were protected from challenge with Lassa virus using a vac-cinia-NC construct, primates were not, but both primates and rodents were effectively vaccinated with vaccinia-GP. Protection was not correlated with antibody level, suggesting a critical role for CMI responses in viral clearance (Auperin et al. 1988; Morrison et al. 1989). A broader study of protection in macaques using a range of recombinant vaccinia expressing NC, GP or combinations of these proteins confirmed the role of GP in protection and the dependence on CMI responses (Fisher-Hoch & McCormick 2001). Unfortunately, vaccinia recombinants are not currently tenable as a vaccine in West Africa because of the potential side effects in an HIV-endemic area. In the longer term, a yellow fever/Lassa fever chimaeric live attenuated virus may offer an attractive solution, although the attenuated strains of vaccinia, MVA and NYVAC, are worthy of consideration.

Formalin-inactivated mouse brain-derived Japanese encephalitis virus (JEV) vaccines are widely used in Asia, and a variety of other inactivated, live-attenuated and chimaeric yellow fever/JEV ('ChimeriVax') vaccines are under development (Monath 2002). Konishi et al. (2001) have developed a stable CHO cell line to express a secreted form of subviral particle (EPs) containing the envelope glycoprotein (E) and a precursor (prM) of the viral membrane protein. EPs were shown to be protective in mice and to have many of the antigenic and biochemical properties of viral E, and may provide a useful source antigen for JEV vaccines and diagnostics. ChimeriVax technology is also being applied to a yellow fever/West Nile virus vaccine, in which the prM and E structural proteins of yellow fever 17D vaccine virus are replaced with the equivalent West Nile genes (Monath 2001).

A vaccine against Ebola virus may help to contain this lethal infection and protect at-risk groups. Trials in a guinea pig model have shown that protection can be conferred using both nucle-oprotein or envelope protein expressed by a Venezuelan equine encephalitis replicon, although the immune correlates of protection remain unclear (Gupta et al. 2001; Pushko et al. 2000; Wilson & Hart 2001). A prime/boost vaccination strategy using a DNA/envelope prime followed by recombinant adenovirus/envelope boost was 100% protective in a small-scale macaque study, but much more work is required to establish the potential of this, or other, Ebola vaccines for human trials (Sullivan et al. 2003). A 2003 trial with this vaccine in humans was unsuccessful (http://www3. Respiratory viruses

The 200-plus serologically distinct viruses transmitted via the respiratory tract are the major cause of community morbidity and hospitalization in the industrialized world, with significant mortality amongst children, the elderly, and those of any age with compromised immune, cardiac or respiratory systems. The majority of respiratory infections are caused by rhinoviruses, but coronaviruses, parainfluenza (PIV), RSV, CMV, AV and influenza viruses A and B also have a major impact (Olszewska et al. 2002). The development of respiratory virus vaccines must take into account the unique clinical and immunological character of these infections. As most infections are localized and mucosal, local secreted antibodies and T-cell responses may be sufficient for protection, and the presence of circulating serum antibody may be just an indication of immunological priming. Also, most respiratory viruses are non-lytic, and the disease is mostly due to inflammatory and immune responses to infection. Thus disease can be potentiated in the presence of inappropriate immune priming, which has been noted with formalin-inactivated RSV viruses (Kim et al. 1969; Openshaw et al. 2001). At present, no vaccines are licensed for use against RSV, PIV, CMV or any rhino- or coronavirus.

Protection from RSV infection is correlated with high titres of mucosal IgA, and a number of vaccines based on the G and F surface proteins are under development. Phase II trials are underway with purified fusion (F) protein obtained from RSV-infected Vero cells and are showing some promise (Piedra et al. 2003). Four different subunit approaches are currently under development. The first employs a fusion protein comprising the major immunodominant domain of the G protein fused to the streptococcal G protein albumin-binding domain. This effectively protects mice against challenge and has entered Phase I trials (Power et al. 2001; Siegrist et al. 1999). The second and third strategies use recombinant viral vaccines. One based on MVA expressing RSV-G revealed promising results in mice, but after showing poor immunogenicity in non-human primates, clinical trials were cancelled (Wyatt et al. 1999). Another viral vector uses bovine RSV successfully to express human RSV-F and -G proteins (Collins et al. 1999) and is immunogenic in mice. Further trials are underway. The fourth, and most novel approach, uses the techniques of reverse genetics, whereby infectious virus is produced entirely from cDNA with mutations introduced so as to attenuate viral pathogenicity while retaining its ability to confer protective immune responses (Murphy & Collins 2002). This technology has been applied to RSV subgroup A strain A2 (Collins et al. 1999), PIV3 virus strain JS (Durbin et al. 1997) and bovine PIV3 (Schmidt et al. 2000). cDNAs from human PIV1 and PIV2 are also now available (Kawano et al. 2001; Newman et al. 2002). Two candidates, based on 4 attenuating genetic elements in the infectious rA2 RSV strains, have been produced as recombinant viruses in Vero cells and used in Phase I/II trials in both adult and infant cohorts. One candidate was found not to be suitable for use in children, given its level of replication in RSV-seronegative patients. However, the other vaccine was well-tolerated in infants, with detectable IgA responses, and vaccinees were protected against a second, homologous challenge. The next stage is to determine whether the vaccine is protective against wild-type viruses (Karron et al. 2005).

Influenza has a notable effect on both the community and individuals. In the USA, up to 20 % of the population may catch 'flu in a given year, and epidemics may cause 20-40 000 deaths. These figures pale into insignificance compared with the great 'flu pandemic of 1918-1919, with millions of deaths worldwide. The epidemiology of 'flu is complicated by the fact that the viruses undergo two forms of antigenic change: antigenic shift, which occurs when genes from animal viruses are transferred to a human virus by reassortment in a dual-infected human host, thus creating a new subtype with novel antigenicity and often associated high morbidity and mortality; and antigenic drift, caused by point mutations within the surface proteins, which may allow immune evasion and disease with time. Two surface proteins are involved in these effects, haemagglutinin (HA) and neuraminidase (NA). Type A viruses are found in both animal reservoirs (primarily birds and swine) and are susceptible to antigenic shift, whereas the type B virus is exclusively human and cannot undergo reassortment. Vaccines must therefore be based on the circulating A virus HA and NA subtypes, plus a B virus component. Continual monitoring of these parameters is necessary, and chemically inactivated viruses, grown in embryonated chicken eggs, are prepared for each year's 'flu season. For this reason, the possibility of producing a 'universal' 'flu vaccine is enticing. Unfortunately, the most conserved regions of the 'flu genome are less immunogenic and less likely to induce a protective response. Nonetheless, a fusion protein consisting of the minor surface antigen M2 from an influenza A virus plus the core protein of hepatitis B provided a high degree of protection against viral challenge in mice (Neirynck et al. 1999), while other trial vaccines based on M2 or NA show strengthened immunogenicity when fused to cytokine genes (Babai et al. 2001; Kilbourne et al. 1995). An alternative approach may be predicting the antigenic trend of evolving viruses and constructing synthetic strains based on a framework of conserved amino acids (Bush et al. 1999). Reverse genetics technology is also being applied, based on the attenuation of the 'flu NS1 protein, which affects viral virulence by modulation of the antiviral IFN response. Preliminary work with mice has shown that IFN-antagonist activity can be down-modulated by truncation of the NS1 gene (Palese & Garcia-Sastre 2002). Prime-boost regimes, based on priming with a DNA-based vaccine followed by a subunit boost may also be worth pursuing, but the immunogenicity of both vaccine components must be optimized to develop worthwhile immunity in the short time-frame available annually (Kemble & Greenberg 2003).

Recently, a serious new infection characterised by severe respiratory system complications ("Severe Acute Respiratory Syndrome" - SARS) emerged in China, leading to serious epidemics in the Far East and Toronto, Canada, causing serious concern regarding international spread. Although not conclusively identified as the agent of all SARS outbreaks, the culprit appears to be a coronavirus (Peiris et al. 2003), and several isolates have now been fully sequenced (Marra et al. 2003; Rota et al. 2003). These are termed SARS-associated coronaviruses (SARS-CoV). The data indicate that the virus is only moderately related to other known coronaviruses. Serological and immunopathological evidence suggests that children only show mild disease manifestations, and clearance of the infection is associated with high titres of circulating antibody to the viral membrane proteins spike (S) and envelope (M). This would suggest that a subunit vaccine might be feasible to control the disease in the longer term, although containment and control measures are the most effective measures in the short term. Currently, SARS vaccine development is adopting three approaches: An inactivated SARS-CoV, a full-length S-protein and a fragment containing the major neutralising epitopes of the S protein (Jiang et al. 2005). The inactivated SARS-CoV vaccine elicited high titres of S-specific antibodies in immunized mice and rabbits that block receptor binding and virus entry (He et al. 2004), and initial results from a Phase I clinical trial in the Guangdong Province of China suggests that this vaccine is safe and induces SARS-CoV-neutralising antibodies (Business Wire, December 6th, 2004) Other viruses

Major efforts are also being directed towards producing improved rabies vaccines. The vaccines currently in use for the immunization of humans and domestic animals are derived from the 'fixed'-type virus of serotype 1/genotype 1, but do not offer protection against other genotypes or bat rabies (Woldehiwet 2002). A vaccinia-rabies recombinant expressing the rabies glycoprotein gene (G) has successfully reduced the incidence of rabies in foxes and other carnivores in Europe (Pastoret & Brochier 1999), increasing the risk of transmission from bat-borne infections (Nadin-Davis et al. 2001). Although rabies is generally controlled by immunization of the animal reservoir, because the disease has a long incubation period in humans it is possible to prevent the development of disease by post-exposure vaccination. Currently, such vaccines are live-attenuated preparations, derived from infected animal brain or cell culture but, due to costs of production and the risk of hypersensitivity, new vaccines are desirable. Viral clearance is strongly associated with both humoral and cell-mediated responses, and is best conferred by a live vaccine. Recombinant G subunit vaccines are ineffective (Chappius 1995). Morimoto et al. (2001) have produced a range of modified rabies virus G genes to engineer rabies recombinant viruses, which exhibit marked decreases in viral infectivity coupled with higher G protein expression than wild-type viruses. Importantly, they are also non-pathogenic in a murine model, but show the highest level of protection when challenged with homologous virus. Immunogenicity may be improved by the use of chimaeric viruses, including fragments of G protein genes from diverse genotypes. DNA vaccination of mice with chimaeric G protein genes from bat, human and canine lyssavirus, conferred protection to heterologous isolates of lyssavirus and indicates that a combined lyssavirus vaccine may be achievable (Jallet et al. 1999).

Rotavirus (RRV) is the most common single cause of severe, dehydrating gastroenteritis worldwide and accounts for 20 % of all diarrhoea deaths in children under 5 years old (de Zoysa & Feachem 1985). At least eight live, attenuated, oral rotavirus vaccines are currently in human trials, but concerns that tetravalent RRV ('Rotashield') may be associated with intussusception in some infants has led to the temporary withdrawal of this particular vaccine (Centre for Disease Control and Protection 1999). However, following additional epidemiological research, this was considered to be an over-reaction to a minor risk, and Rotashield has now been re-licensed (Murphy et al. 2003). Alternative vaccine designs based on the use of virus-like particles as subunit vaccines are currently being pursued in animal models, but are at a very early stage of development (Cunliffe et al. 2002).

Parvovirus B19 is the only member of the family Parvoviridae known to be pathogenic in humans, manifestations of infection ranging from a mild rash to fetal death in utero (Heegaard & Brown 2002). An empty B19 VP1/VP2 VLP expressed in baculovirus is potently immunogenic in a number of experimental animals, VP1 inducing a high-titre neutralizing antibody response in human volunteers. Phase I trials produced encouraging results and Phase II trials are now underway (Bansal et al. 1993; Bostic et al. 1999; Kajigaya et al. 1991).

There is growing evidence that SV40 may be involved in the development of certain human cancers and Phase I trials with a recombinant vaccinia expressing modified large T antigen are now underway (Imperiale et al. 2001).

Effective live, attenuated virus vaccines are available for the four major viral diseases of childhood, measles (MV), mumps (paramyxovirus - PMV), rubella (RV) and varicella (VZV) and are in worldwide use in various combinations. The benefits of childhood vaccination with the combined measles-mumps-rubella (MMR) vaccine have been clearly demonstrated in the UK and elsewhere and no credible scientific evidence has yet substantiated the claims that MMR vaccine causes Crohn's disease or autism (Miller 2002). Indeed, a tetravalent vaccine including varicella (MMRV) is now proposed for use (Nolan et al. 2002). Despite this, novel vaccines are still required. Live attenuated measles vaccines are ineffective in infants aged <6-9 months (Redd et al. 1999) leading to significant worldwide mortality in children aged under 12 months, and there are doubts as to the effectiveness of the mumps virus component (Pons et al. 2000). A number of live viral vectors expressing the dominant measles haemagglutinin (MV-HA) antigen, which contains both B- and T-cell epitopes, are currently under examination in both rodent and nonhuman primate models. These include replication-deficient vectors such as ALVAC (Taylor et al. 1992), MVA (Stittelaar et al. 2000), NYVAC (Kovarik et al. 2001) and AV (Fooks et al. 1998) and replication-competent vectors such as VSV (Schlereth et al. 2000) and PIV3 (Durbin et al. 2000). The challenge for such vaccines is to induce adult-like antibodies, Th1-like and CTL responses in infants. Infant mice respond well to the NYVAC-HA vaccine, but not to ALVAC-HA, despite both encoding the same antigen and showing identical T-cell responses in adult mice (Kovarik et al. 2001). Unfortunately, mice cannot be used for challenge studies, and optimal strategies, possibly based on prime-boost regimes will have to be undertaken in macaques before promising modalities are transferred to Phase I/II human trials.

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