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Immunogens of encephalitis viruses
Veterinary Microbiology, 37 (1993) 273-284 Elsevier Science Publishers B.V., Amsterdam
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Immunogens of encephalitis viruses John T. Roehrig Division of Vector-BorneInfectious Diseases, National Centers for Infectious Diseases, Centers for Disease Control, Public Health Service, U.S. Department of Health and Human Services, Fort Collins, CO, USA (Accepted 14 July 1993)
ABSTRACT The equine encephalitis viruses are members of the genus Alphavirus, in the family Togaviridae. Three main virus serogroups represented by western (WEE), eastern (EEE) and Venezuelan equine encephalitis (VEE) viruses cause epizootic and enzootic infection of horses throughout the western hemisphere. All equine encephalitis viruses are transmitted through the bite of an infected mosquito. The first equine encephalitis virus vaccines were produced by virus inactivation. Problems with inadequate inactivation,which may have caused a major epidemic/epizootic of VEE in central America and Texas in the 1970s, led to the development of a live attenuated VEE virus vaccine (TC-83) derived by cell culture passage. Inactivated vaccines are still used to prevent equine infections with WEE and EEE viruses. Alphaviruses are small single stranded, positive sense RNA viruses. The 12000 nucleotide genome is enclosed in an icosahedral nucleocapsid composed of multiple copies of the capsid (C) protein. The virion is enveloped. The membrane is modified by the insertion of heterodimers of two glycoproteins: E 1 and E2. Monoclonal antibody analysis of the surface glycoproteins have provided a detailed understanding of important protective antigens. Recent studies comparing gene sequences from virulent and avirulent VEE viruses have begun to delineate mechanisms of alphavirus attenuation.
Alphaviruses, members of the family Togaviridae, are the primary viral agents of encephalitic infections of veterinary importance (Monath and Trent, 1981 ). Three serocomplexes represented by eastern, western and Venezuelan equine encephalitis (EEE, WEE and VEE) viruses are established in the western hemisphere (Table 1 ). The EEE virus serocomplex can be further divided into North American and South American subtypes (Roehrig et al., 1990). These subtypes do not apparently cocirculate, and are restricted to their continent of origin (Casals, 1964; Calisher et al., 1971 ). The WEE virus serocomplex contains a number of antigenically related viruses. Highlands J viCorrespondence to: J.T. Roehrig, Division of Vector-Borne Infectious Diseases, National Centers for Infectious Diseases, Centers for Disease Control, Public Health Service, U.S. Department of Health and Human Services, P.O. Box 2087, Fort Collins, Colorado, 80522, USA.
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TABLE 1 Important alphavirus disease agents of the western hemisphere Complex
Virus prototype
Subtypes, variants or related viruses
Western equine Eastern equine
WEE EEE
Venezuelan equine
VEE
Highlands J North American EEE South American EEE Epizootic subtypes (lAB, 1C) Enzootic subtypes (1D, 1E, IF, 2, 3, 4, 5, 6)
rus, a relatively non-pathogenic alphavirus, is of clinical importance because it cocirculates with EEE virus and causes confusion in EEE virus laboratory diagnosis (Karabatsos, et al. 1988). VEE viruses have been isolated from the southern United States, central America and South America. The VEE virus serocomplex can also be divided into a number of subtypes and varieties, based upon serological reactivities (Young and Johnson, 1969). Serologic classification of VEE viruses is a useful tool in predicting the epizootic potential of VEE virus isolates. Alphaviral encephalitis is usually epizootic or epidemic in nature which means that the appearance of serious disease is infrequent (Shope 1985 ). In many cases, however, enzootic transmission cycles can be maintained. The contribution of these enzootic cycles to epizootic or epidemic disease is not known. Alphavirus infection is initiated by the bite of an infected mosquito. Each of these viruses are maintained in nature in their own host reservoirmosquito vector cycles (for review see Tsai and Monath, 1987 ). Because encephalitis is a severe disease in nonimmunized animals, virus transmission to the equine or human populations is a serious veterinary and public health concern.
Alphavirus structure and replication Much of the information presented in this section has been derived from studies with less pathogenic, laboratory adapted alphaviruses, such as Sindbis (SIN) virus and Semliki Forest (SF) virus (for more detailed review see Strauss and Strauss, 1986). The conclusions derived from these investigations appear to be valid for the more pathogenic WEE, EEE and VEE viruses and will be cited when appropriate. During alphavirus replication a number ofnonstructural proteins are synthesized. Because solid immunity can be elicited by immunization with the envelope proteins, this short review will focus on the envelope proteins and their contribution to alphavirus immunology. Alphaviruses are small positive stranded RNA viruses. The genome is approximately 12000 nucleotides in length. Genomic sequences are known for
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six alphaviruses (Garoff et al., 1980; Rice and Strauss, 1981; Dalgarno et al., 1983; Kinney et al., 1986; 1989; Chang and Trent 1987; Hahn et al., 1988). The virion RNA is enclosed in a nucleocapsid comprised of multiple copies of a core or capsid protein (C, 35 kDa). The virion is enveloped, and the envelope, which is derived from host cell membranes, is modified by the insertion of two virus specific envelope glycoproteins: El, 50-55 kDa, and E2, 50-55 kDa. There is approximate equimolar distribution of the C, E1 and E2 in the virion. The E1 and E2 are expressed as heterodimers, and three of these dimers associate to form the spikes observed on the virion surface (Fuller 1987). Little is known about the exact physical interaction of these two proteins. Virus infection appears to be initiated by attachment to plasma membrane receptors. Recent studies have implicated various proteins as attachment sites for SIN virus (Ubol and Griffin, 1991: Wang et al., 1991 ). Preincubation of virus with virus neutralizing monoclonal antibodies (mAbs) specific for both E2 and E 1 of VEE virus blocks virus attachment (Roehrig et al., 1988 ). Following attachment to cellular receptor the virus presumably enters endocytic vesicles (for review see Kielan and Helenius, 1986). The low pH of the endocytic vesicle causes a conformational shift in the structure of one or both of the envelope glycoproteins and expresses the membrane fusion sequence located on the E 1 protein (Boggs et al., 1989; Levy-Mintz and Kielan, 1991 ). The release of the nucleocapsid into the cytoplasm is presumably mediated by the fusion of the virus envelope with the membrane of the endocytic vesicle. The viralstructural proteins are translated as a polyprotein from a 26S subgenomic messenger RNA derived from the 3'-one third of the genome (Strauss and Strauss, 1986). The complete nucleotide sequence of the 26S RNA has been published for a number of alphaviruses. The nucleocapsid is synthesized first and is autocatalytically cleaved from the nascent polypeptide chain (Aliperti and Schlesinger, 1978; Choi et al., 1991 ). The E2 is synthesized next in the form of a 65 kDa precursor protein, PE2. The non-E2 portion of the PE2 probably functions as a PE2 signal sequence directing PE2 insertion into the exocytic vesicle membranes of the Golgi (Schlesinger and Schlesinger, 1986). Following PE2 insertion and cleavage from the nascent polypeptide chain, the 6 kDa and E1 proteins are synthesized. The 6 kDa protein is located before the E 1 amino terminus and probably functions as the E 1 signal sequence. Recent investigations indicate that this 6 kDa protein is maintained in the SIN virus envelope but not in the SF virus envelope (Gaedigk-Nitschko and Schlesinger, 1990; Lusa et al., 1991 ). The PE2 and the E 1 are cotranslationally glycosylated and rapidly associate into heterodimers. The PE2 to E2 cleavage occurs later in replication. Investigations with SIN virus suggest that PE2 cleavage may be necessary for replication in the mosquito host only (Presley et al., 1991 ). The effect that a failed PE2 to E2 cleavage has on subsequent attachment to cellular receptor is not known.
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Antigenic structure of the E2 and E1 glycoprotein Early studies with SIN virus using polyclonal but monospecific antisera raised against native E 1 or E2 assigned the biological function of elicitation of virus neutralizing antibodies to the E2 glycoprotein, and elicitation of hemagglutination inhibiting antibodies to the E 1 glycoprotein (Dalrymple et al., 1976). These original observations were confirmed later in studies with WEE and VEE viruses (Trent and Grant, 1980; France et al., 1979; Kinney et al., 1983). A more detailed understanding of the immunological and biological characteristics of the E2 and E1 glycoproteins has been derived from studies using mAbs (for detailed reviews see Roehrig, 1986; Roehrig, 1990). As with other protein antigens, multiple epitopes can be identified on both the E2 and E1 glycoproteins. Both glycoproteins are able to elicit virus neutralizing antibody, however the neutralizing antibody elicited by the E2 is quantitatively more potent and protective (Mathews and Roehrig, 1982; Roehrig et al., 1982; Roehrig et al., 1985 ). By determining the amino acid sequences of variants resistant to neutralization by mAbs, an E2 neutralization domain has been identified between amino acids 180 and 220 on SIN and VEE viruses (Stec et al., 1986; Strauss et al., 1987; Johnson et al., 1990; Strauss et al., 1991 ) (Table 2 ). A similar domain is located slightly closer to the carboxyl terminus (amino acids 216-251 ) of Ross River (RR) virus E2 glycoprotein (Vrati et al. 1988 ). Expression of this neutralization domain appears to have a strong conformational component because some of the binding capacity of the E2 specific neutralizing mAbs is lost following reduction and alkylation of the virus, and because synthetic peptides prepared from the deduced amino acid sequence of the E2 neutralization domain fail to elicit neutralizing antibody or bind neutralizing E2 glycoprotein specific mAbs (Johnson et al., 1990; Hunt et al., 1990). Virus neutralizing activity can also be associated with E 1 glycoprotein speTABLE2 Alphavirus protective domains Virus
Position
Method
Reference
VEE
E2 182-209 E2 1-19 E2 241-265 E1 132 E2 181-216 E1 132 E2 216-251 E2 289-352 E2 240-255
mab NT Escape Synthetic peptide Synthetic peptide mab NT Escape mab NT Escape mab NT Escape mab NT escape Gene expression Synthetic peptide
Johnson et al., (1990) Hunt et al., (1990) Johnson et al., (1990) Roehrig (unpublished) Strauss et al., (1987) Strauss et al., ( 1991 ) Vrati et al., (1988) Grosfeld et al., (1989) Snijders et al., ( 1991 )
SIN RR SF
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cific mAbs (Schmaljohn et al., 1982; 1983; Mathews and Roehrig, 1982; Boere et al., 1984; Stanley et al., 1986; Mendoza et al., 1988). Sequencing of E1 mAb neutralization escape variants of SIN and VEE viruses identify changes at amino acid 132 (Strauss et al., 1991; JTR unpublished observation). In the case of VEE virus the involvement of E 1 epitopes in virus neutralization is believed to be linked to the close spatial association of these epitopes with the E2 neutralization domain (Roehrig et al., 1982). The VEE virus E1 glycoprotein neutralization epitope appears to be more linear in nature than its E2 glycoprotein counterpart because it is stable to reduction and alkylation (JTR unpublished observation). A synthetic peptide prepared from this deduced E 1 sequence, however, also fails to elicit neutralizing antibody. Neutralization activity has been associated with other E 1 glycoprotein epitopes on SIN, SF and VEE viruses (Schmaljohn et al., 1982; Mathews and Roehrig, 1982). Many of these epitopes are shared by all alphaviruses and appear to have a large conformational component because while they are accessible on the surface of the infected cell they are hidden or cryptic on the surface of the mature virion (Schmaljohn et al., 1982; 1983; Hunt and Roehrig, 1985 ). Detergent disruption of the virus exposes these epitopes. Because mAbs specific for these cryptic epitopes also function in blocking the agglutination or lysis of erythrocytes it is possible that these antibodies could neutralize virus infectivity by blocking virus mediated membrane fusion.
Protective epitopes on the alphavirus E2 and E1 glycoproteins For alphaviruses, solid protection from virus infection can be accomplished by eliciting virus neutralizing antibody. It is not surprising, therefore, that immunization with subunit vaccines that elicit neutralizing antibody (e.g. pure native E2 glycoprotein) is protective. It is also not surprising that passive transfer of neutralizing mAbs to nonimmune animals results in solid protection from infection (Mathews and Roehrig, 1982; Roehrig et al., 1985 ). Recent data suggests, however, that antibody mediated protection can also result from immunization with synthetic peptides or bacterially expressed subunit peptides that do not elicit virus neutralizing antibodies (Grosfeld et al., 1989; Hunt et al., 1990; Hunt et al., 1991; Johnson et al., 1991; Snijders et al., 1991 ). It has been shown that a synthetic peptide derived from the first 25 amino acids of the VEE virus E2 glycoprotein (VE2pep01/Trd) can confer solid protection to outbred mice. Passive transfer of polyclonal or monoclonal anti-VE2pep01/Trd can also protect nonimmune mice. While the molecular mechanism of protection has not been well defined, the antipeptide appears to attenuate the virus infection by limiting in vivo virus replication until the host immune response aborts the infection. As with all protein antigens there is also a T-cell component associated with virus infection. In its simplest interpretation, T helper (Tn) cells must be activated for proper antibody response. The breadth of the Th-cell response has
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been analyzed with RR, WEE, EEE and VEE viruses (Aaskov et al., 1983; Mathews et al., 1989). This response appears to be complex specific in the primary infection, but broadens with multiple exposures. The contribution of each envelope glycoprotein to activation of Th-cells is currently being investigated (J. Mathews, personal communication). Detailed investigations on the induction of alphavirus specific cytotoxic Tcells (To-cells) have been reported (Mullbacher and Blanden, 1978; Mullbacher et al., 1979; Wolcott et al., 1984 ). There appears to be epitopes located on the E1 glycoprotein capable of eliciting alphavirus crossreactive protective Tc-ceUs. The contribution of To-cells to protection from infection is not fully understood.
Quantitation of protective immunogens There are currently two types of protective alphavirus immunogens that require quantitative assays. The first type is inactivated immunogens such as subunit immunogens that consist of some combination of the E 1 and E2 glycoproteins, or inactivated virus immunogens. Because of the availability of glycoprotein specific mAbs, differential quantitation of E 1 and E2 can be performed in standard indirect ELISA format for VEE virus antigens (Roehrig, 1986). Only mAbs specific for the E 1 glycoproteins of EEE and WEE viruses are currently available. The highly conformational character of the E 1, and to a lesser extent the E2, makes attention to immunogen isolation procedures imperative. The second type of protective immunogen is live attenuated virus. These immunogens can also be tested in standard indirect ELISA or antigen capture ELISA. The availability of mAbs which bind in ELISA to important virus epitopes allows for rapid screening of new vaccine candidates for the presence of protective epitopes. The potency of live vaccines ultimately depends on their replication efficacy and immunogenicity. The only way to test these characteristics of new immunogen candidates, is still inoculation of animal models.
Current alphaviruses vaccine strategies Protection of horses from infection with EEE and WEE viruses is currently provided by immunization with inactivated virus vaccines derived from infected cell culture. Three VEE virus vaccines currently exist. One VEE vaccine is a live vaccine (TC-83) that was attenuated by passage in cell culture (Berge et al., 1961 ). A formalin inactivated derivative of TC-83 (C84) has also been prepared and characterized (Cole et al., 1974). A more recent development has been the expression of the VEE virus structural genes in vaccinia virus. This construct has been shown to be protective for mice, equines and primates (Kinney et al., 1988a; 1988b; Bowen et al., 1992; Monath et al., 1992).
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USDA requirements for safety testing of the inactivated EEE and WEE vaccines requires subcutaneous virulence testing in 6 to 12 h old chickens (Anonymous, 1974). VEE vaccine safety testing is proprietary, but usually includes virulence testing in small animal models such as mice or guinea pigs followed by inoculation of horses. Potency testing for bulk or final samples of killed EEE and WEE virus vaccines is a two stage process involving inoculation of guinea pigs and analysis of the subsequent antibody titer by plaque reduction neutralization tests (PRNT) with the homologous virus.
Future alphavirus vaccine development The major drawback to currently available inactivated EEE, WEE and VEE virus vaccines is the short duration of immunity. As with other viruses, long lasting immunity is best elicited by a live attenuated virus vaccine. The attenuated VEE virus vaccine, TC-83, gives longer lasting immunity, however, it suffers from reactogenicity in some individuals and nonimmunogenicity in others. The nonimmunogenic features of some vaccines may ultimately be overcome with a better understanding of the T-cell response. A number of investigations have begun to unravel various mechanisms of alphavirus attenuation. An amino acid change in the E2 of SIN and VEE viruses results in rapid penetrating mutants that are attenuated in animals (Olmsted et al., 1984; 1986; Davis et al., 1986; 1991; Johnston and Smith, 1988 ). A comparison of the sequence of the structural gene region of a wild type, virulent VEE virus and its vaccine derivative, revealed only six amino acid changes, 5 in the E2 and one in the E1 (Johnson et al., 1986; Kinney et al., 1989). It is clear from these results that stable alphavirus attenuation may require only a few amino acid mutations. A most promising advance in alphavirus vaccine design has been the preparation of infectious, full-length cDNA cloned virus, derived from the viral RNA (Davis et al., 1989; 1991; Kinney et al., 1992). This development will allow for detailed analysis of virus virulence and pathogenicity by using site directed mutagenesis to create and test the attenuating potential and stability of any possible genetic change. Storing the genes of the vaccine strain in the form of DNA will allow for stable maintenance of the attenuated phenotype. Continued progress in molecular analysis of alphavirus attenuation and antigenicity will most certainly produce potent and stable second generation alphavirus vaccines.
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cytolysis of cells infected with a temperature-sensitive mutant of Sindbis virus. J. Gen. Virol., 66:1167-1170. Young, N.A., and Johnson, K.M., 1969. Antigenic variants of Venezuelan equine encephalomyelitis virus: Their geographic distribution and epidemiologic significance. Am. J. Epidemiol., 89: 286-307.
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Immunogens of encephalitis viruses John T. Roehrig Division of Vector-BorneInfectious Diseases, National Centers for Infectious Diseases, Centers for Disease Control, Public Health Service, U.S. Department of Health and Human Services, Fort Collins, CO, USA (Accepted 14 July 1993)
ABSTRACT The equine encephalitis viruses are members of the genus Alphavirus, in the family Togaviridae. Three main virus serogroups represented by western (WEE), eastern (EEE) and Venezuelan equine encephalitis (VEE) viruses cause epizootic and enzootic infection of horses throughout the western hemisphere. All equine encephalitis viruses are transmitted through the bite of an infected mosquito. The first equine encephalitis virus vaccines were produced by virus inactivation. Problems with inadequate inactivation,which may have caused a major epidemic/epizootic of VEE in central America and Texas in the 1970s, led to the development of a live attenuated VEE virus vaccine (TC-83) derived by cell culture passage. Inactivated vaccines are still used to prevent equine infections with WEE and EEE viruses. Alphaviruses are small single stranded, positive sense RNA viruses. The 12000 nucleotide genome is enclosed in an icosahedral nucleocapsid composed of multiple copies of the capsid (C) protein. The virion is enveloped. The membrane is modified by the insertion of heterodimers of two glycoproteins: E 1 and E2. Monoclonal antibody analysis of the surface glycoproteins have provided a detailed understanding of important protective antigens. Recent studies comparing gene sequences from virulent and avirulent VEE viruses have begun to delineate mechanisms of alphavirus attenuation.
Alphaviruses, members of the family Togaviridae, are the primary viral agents of encephalitic infections of veterinary importance (Monath and Trent, 1981 ). Three serocomplexes represented by eastern, western and Venezuelan equine encephalitis (EEE, WEE and VEE) viruses are established in the western hemisphere (Table 1 ). The EEE virus serocomplex can be further divided into North American and South American subtypes (Roehrig et al., 1990). These subtypes do not apparently cocirculate, and are restricted to their continent of origin (Casals, 1964; Calisher et al., 1971 ). The WEE virus serocomplex contains a number of antigenically related viruses. Highlands J viCorrespondence to: J.T. Roehrig, Division of Vector-Borne Infectious Diseases, National Centers for Infectious Diseases, Centers for Disease Control, Public Health Service, U.S. Department of Health and Human Services, P.O. Box 2087, Fort Collins, Colorado, 80522, USA.
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TABLE 1 Important alphavirus disease agents of the western hemisphere Complex
Virus prototype
Subtypes, variants or related viruses
Western equine Eastern equine
WEE EEE
Venezuelan equine
VEE
Highlands J North American EEE South American EEE Epizootic subtypes (lAB, 1C) Enzootic subtypes (1D, 1E, IF, 2, 3, 4, 5, 6)
rus, a relatively non-pathogenic alphavirus, is of clinical importance because it cocirculates with EEE virus and causes confusion in EEE virus laboratory diagnosis (Karabatsos, et al. 1988). VEE viruses have been isolated from the southern United States, central America and South America. The VEE virus serocomplex can also be divided into a number of subtypes and varieties, based upon serological reactivities (Young and Johnson, 1969). Serologic classification of VEE viruses is a useful tool in predicting the epizootic potential of VEE virus isolates. Alphaviral encephalitis is usually epizootic or epidemic in nature which means that the appearance of serious disease is infrequent (Shope 1985 ). In many cases, however, enzootic transmission cycles can be maintained. The contribution of these enzootic cycles to epizootic or epidemic disease is not known. Alphavirus infection is initiated by the bite of an infected mosquito. Each of these viruses are maintained in nature in their own host reservoirmosquito vector cycles (for review see Tsai and Monath, 1987 ). Because encephalitis is a severe disease in nonimmunized animals, virus transmission to the equine or human populations is a serious veterinary and public health concern.
Alphavirus structure and replication Much of the information presented in this section has been derived from studies with less pathogenic, laboratory adapted alphaviruses, such as Sindbis (SIN) virus and Semliki Forest (SF) virus (for more detailed review see Strauss and Strauss, 1986). The conclusions derived from these investigations appear to be valid for the more pathogenic WEE, EEE and VEE viruses and will be cited when appropriate. During alphavirus replication a number ofnonstructural proteins are synthesized. Because solid immunity can be elicited by immunization with the envelope proteins, this short review will focus on the envelope proteins and their contribution to alphavirus immunology. Alphaviruses are small positive stranded RNA viruses. The genome is approximately 12000 nucleotides in length. Genomic sequences are known for
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six alphaviruses (Garoff et al., 1980; Rice and Strauss, 1981; Dalgarno et al., 1983; Kinney et al., 1986; 1989; Chang and Trent 1987; Hahn et al., 1988). The virion RNA is enclosed in a nucleocapsid comprised of multiple copies of a core or capsid protein (C, 35 kDa). The virion is enveloped, and the envelope, which is derived from host cell membranes, is modified by the insertion of two virus specific envelope glycoproteins: El, 50-55 kDa, and E2, 50-55 kDa. There is approximate equimolar distribution of the C, E1 and E2 in the virion. The E1 and E2 are expressed as heterodimers, and three of these dimers associate to form the spikes observed on the virion surface (Fuller 1987). Little is known about the exact physical interaction of these two proteins. Virus infection appears to be initiated by attachment to plasma membrane receptors. Recent studies have implicated various proteins as attachment sites for SIN virus (Ubol and Griffin, 1991: Wang et al., 1991 ). Preincubation of virus with virus neutralizing monoclonal antibodies (mAbs) specific for both E2 and E 1 of VEE virus blocks virus attachment (Roehrig et al., 1988 ). Following attachment to cellular receptor the virus presumably enters endocytic vesicles (for review see Kielan and Helenius, 1986). The low pH of the endocytic vesicle causes a conformational shift in the structure of one or both of the envelope glycoproteins and expresses the membrane fusion sequence located on the E 1 protein (Boggs et al., 1989; Levy-Mintz and Kielan, 1991 ). The release of the nucleocapsid into the cytoplasm is presumably mediated by the fusion of the virus envelope with the membrane of the endocytic vesicle. The viralstructural proteins are translated as a polyprotein from a 26S subgenomic messenger RNA derived from the 3'-one third of the genome (Strauss and Strauss, 1986). The complete nucleotide sequence of the 26S RNA has been published for a number of alphaviruses. The nucleocapsid is synthesized first and is autocatalytically cleaved from the nascent polypeptide chain (Aliperti and Schlesinger, 1978; Choi et al., 1991 ). The E2 is synthesized next in the form of a 65 kDa precursor protein, PE2. The non-E2 portion of the PE2 probably functions as a PE2 signal sequence directing PE2 insertion into the exocytic vesicle membranes of the Golgi (Schlesinger and Schlesinger, 1986). Following PE2 insertion and cleavage from the nascent polypeptide chain, the 6 kDa and E1 proteins are synthesized. The 6 kDa protein is located before the E 1 amino terminus and probably functions as the E 1 signal sequence. Recent investigations indicate that this 6 kDa protein is maintained in the SIN virus envelope but not in the SF virus envelope (Gaedigk-Nitschko and Schlesinger, 1990; Lusa et al., 1991 ). The PE2 and the E 1 are cotranslationally glycosylated and rapidly associate into heterodimers. The PE2 to E2 cleavage occurs later in replication. Investigations with SIN virus suggest that PE2 cleavage may be necessary for replication in the mosquito host only (Presley et al., 1991 ). The effect that a failed PE2 to E2 cleavage has on subsequent attachment to cellular receptor is not known.
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Antigenic structure of the E2 and E1 glycoprotein Early studies with SIN virus using polyclonal but monospecific antisera raised against native E 1 or E2 assigned the biological function of elicitation of virus neutralizing antibodies to the E2 glycoprotein, and elicitation of hemagglutination inhibiting antibodies to the E 1 glycoprotein (Dalrymple et al., 1976). These original observations were confirmed later in studies with WEE and VEE viruses (Trent and Grant, 1980; France et al., 1979; Kinney et al., 1983). A more detailed understanding of the immunological and biological characteristics of the E2 and E1 glycoproteins has been derived from studies using mAbs (for detailed reviews see Roehrig, 1986; Roehrig, 1990). As with other protein antigens, multiple epitopes can be identified on both the E2 and E1 glycoproteins. Both glycoproteins are able to elicit virus neutralizing antibody, however the neutralizing antibody elicited by the E2 is quantitatively more potent and protective (Mathews and Roehrig, 1982; Roehrig et al., 1982; Roehrig et al., 1985 ). By determining the amino acid sequences of variants resistant to neutralization by mAbs, an E2 neutralization domain has been identified between amino acids 180 and 220 on SIN and VEE viruses (Stec et al., 1986; Strauss et al., 1987; Johnson et al., 1990; Strauss et al., 1991 ) (Table 2 ). A similar domain is located slightly closer to the carboxyl terminus (amino acids 216-251 ) of Ross River (RR) virus E2 glycoprotein (Vrati et al. 1988 ). Expression of this neutralization domain appears to have a strong conformational component because some of the binding capacity of the E2 specific neutralizing mAbs is lost following reduction and alkylation of the virus, and because synthetic peptides prepared from the deduced amino acid sequence of the E2 neutralization domain fail to elicit neutralizing antibody or bind neutralizing E2 glycoprotein specific mAbs (Johnson et al., 1990; Hunt et al., 1990). Virus neutralizing activity can also be associated with E 1 glycoprotein speTABLE2 Alphavirus protective domains Virus
Position
Method
Reference
VEE
E2 182-209 E2 1-19 E2 241-265 E1 132 E2 181-216 E1 132 E2 216-251 E2 289-352 E2 240-255
mab NT Escape Synthetic peptide Synthetic peptide mab NT Escape mab NT Escape mab NT Escape mab NT escape Gene expression Synthetic peptide
Johnson et al., (1990) Hunt et al., (1990) Johnson et al., (1990) Roehrig (unpublished) Strauss et al., (1987) Strauss et al., ( 1991 ) Vrati et al., (1988) Grosfeld et al., (1989) Snijders et al., ( 1991 )
SIN RR SF
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cific mAbs (Schmaljohn et al., 1982; 1983; Mathews and Roehrig, 1982; Boere et al., 1984; Stanley et al., 1986; Mendoza et al., 1988). Sequencing of E1 mAb neutralization escape variants of SIN and VEE viruses identify changes at amino acid 132 (Strauss et al., 1991; JTR unpublished observation). In the case of VEE virus the involvement of E 1 epitopes in virus neutralization is believed to be linked to the close spatial association of these epitopes with the E2 neutralization domain (Roehrig et al., 1982). The VEE virus E1 glycoprotein neutralization epitope appears to be more linear in nature than its E2 glycoprotein counterpart because it is stable to reduction and alkylation (JTR unpublished observation). A synthetic peptide prepared from this deduced E 1 sequence, however, also fails to elicit neutralizing antibody. Neutralization activity has been associated with other E 1 glycoprotein epitopes on SIN, SF and VEE viruses (Schmaljohn et al., 1982; Mathews and Roehrig, 1982). Many of these epitopes are shared by all alphaviruses and appear to have a large conformational component because while they are accessible on the surface of the infected cell they are hidden or cryptic on the surface of the mature virion (Schmaljohn et al., 1982; 1983; Hunt and Roehrig, 1985 ). Detergent disruption of the virus exposes these epitopes. Because mAbs specific for these cryptic epitopes also function in blocking the agglutination or lysis of erythrocytes it is possible that these antibodies could neutralize virus infectivity by blocking virus mediated membrane fusion.
Protective epitopes on the alphavirus E2 and E1 glycoproteins For alphaviruses, solid protection from virus infection can be accomplished by eliciting virus neutralizing antibody. It is not surprising, therefore, that immunization with subunit vaccines that elicit neutralizing antibody (e.g. pure native E2 glycoprotein) is protective. It is also not surprising that passive transfer of neutralizing mAbs to nonimmune animals results in solid protection from infection (Mathews and Roehrig, 1982; Roehrig et al., 1985 ). Recent data suggests, however, that antibody mediated protection can also result from immunization with synthetic peptides or bacterially expressed subunit peptides that do not elicit virus neutralizing antibodies (Grosfeld et al., 1989; Hunt et al., 1990; Hunt et al., 1991; Johnson et al., 1991; Snijders et al., 1991 ). It has been shown that a synthetic peptide derived from the first 25 amino acids of the VEE virus E2 glycoprotein (VE2pep01/Trd) can confer solid protection to outbred mice. Passive transfer of polyclonal or monoclonal anti-VE2pep01/Trd can also protect nonimmune mice. While the molecular mechanism of protection has not been well defined, the antipeptide appears to attenuate the virus infection by limiting in vivo virus replication until the host immune response aborts the infection. As with all protein antigens there is also a T-cell component associated with virus infection. In its simplest interpretation, T helper (Tn) cells must be activated for proper antibody response. The breadth of the Th-cell response has
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been analyzed with RR, WEE, EEE and VEE viruses (Aaskov et al., 1983; Mathews et al., 1989). This response appears to be complex specific in the primary infection, but broadens with multiple exposures. The contribution of each envelope glycoprotein to activation of Th-cells is currently being investigated (J. Mathews, personal communication). Detailed investigations on the induction of alphavirus specific cytotoxic Tcells (To-cells) have been reported (Mullbacher and Blanden, 1978; Mullbacher et al., 1979; Wolcott et al., 1984 ). There appears to be epitopes located on the E1 glycoprotein capable of eliciting alphavirus crossreactive protective Tc-ceUs. The contribution of To-cells to protection from infection is not fully understood.
Quantitation of protective immunogens There are currently two types of protective alphavirus immunogens that require quantitative assays. The first type is inactivated immunogens such as subunit immunogens that consist of some combination of the E 1 and E2 glycoproteins, or inactivated virus immunogens. Because of the availability of glycoprotein specific mAbs, differential quantitation of E 1 and E2 can be performed in standard indirect ELISA format for VEE virus antigens (Roehrig, 1986). Only mAbs specific for the E 1 glycoproteins of EEE and WEE viruses are currently available. The highly conformational character of the E 1, and to a lesser extent the E2, makes attention to immunogen isolation procedures imperative. The second type of protective immunogen is live attenuated virus. These immunogens can also be tested in standard indirect ELISA or antigen capture ELISA. The availability of mAbs which bind in ELISA to important virus epitopes allows for rapid screening of new vaccine candidates for the presence of protective epitopes. The potency of live vaccines ultimately depends on their replication efficacy and immunogenicity. The only way to test these characteristics of new immunogen candidates, is still inoculation of animal models.
Current alphaviruses vaccine strategies Protection of horses from infection with EEE and WEE viruses is currently provided by immunization with inactivated virus vaccines derived from infected cell culture. Three VEE virus vaccines currently exist. One VEE vaccine is a live vaccine (TC-83) that was attenuated by passage in cell culture (Berge et al., 1961 ). A formalin inactivated derivative of TC-83 (C84) has also been prepared and characterized (Cole et al., 1974). A more recent development has been the expression of the VEE virus structural genes in vaccinia virus. This construct has been shown to be protective for mice, equines and primates (Kinney et al., 1988a; 1988b; Bowen et al., 1992; Monath et al., 1992).
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USDA requirements for safety testing of the inactivated EEE and WEE vaccines requires subcutaneous virulence testing in 6 to 12 h old chickens (Anonymous, 1974). VEE vaccine safety testing is proprietary, but usually includes virulence testing in small animal models such as mice or guinea pigs followed by inoculation of horses. Potency testing for bulk or final samples of killed EEE and WEE virus vaccines is a two stage process involving inoculation of guinea pigs and analysis of the subsequent antibody titer by plaque reduction neutralization tests (PRNT) with the homologous virus.
Future alphavirus vaccine development The major drawback to currently available inactivated EEE, WEE and VEE virus vaccines is the short duration of immunity. As with other viruses, long lasting immunity is best elicited by a live attenuated virus vaccine. The attenuated VEE virus vaccine, TC-83, gives longer lasting immunity, however, it suffers from reactogenicity in some individuals and nonimmunogenicity in others. The nonimmunogenic features of some vaccines may ultimately be overcome with a better understanding of the T-cell response. A number of investigations have begun to unravel various mechanisms of alphavirus attenuation. An amino acid change in the E2 of SIN and VEE viruses results in rapid penetrating mutants that are attenuated in animals (Olmsted et al., 1984; 1986; Davis et al., 1986; 1991; Johnston and Smith, 1988 ). A comparison of the sequence of the structural gene region of a wild type, virulent VEE virus and its vaccine derivative, revealed only six amino acid changes, 5 in the E2 and one in the E1 (Johnson et al., 1986; Kinney et al., 1989). It is clear from these results that stable alphavirus attenuation may require only a few amino acid mutations. A most promising advance in alphavirus vaccine design has been the preparation of infectious, full-length cDNA cloned virus, derived from the viral RNA (Davis et al., 1989; 1991; Kinney et al., 1992). This development will allow for detailed analysis of virus virulence and pathogenicity by using site directed mutagenesis to create and test the attenuating potential and stability of any possible genetic change. Storing the genes of the vaccine strain in the form of DNA will allow for stable maintenance of the attenuated phenotype. Continued progress in molecular analysis of alphavirus attenuation and antigenicity will most certainly produce potent and stable second generation alphavirus vaccines.
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