Kathleen L. Coelingh

Principles underlying the development and use of live attenuated cold-adapted influenza A and B virus vaccines.

THE COLD-ADAPTED (ca) influenza A and B viruses developed by Maassab show great promise for use as a live attenuated virus vaccine to protect against the upper and lower respiratory tract disease caused by the influenza viruses (8,52,58). Since influenza A and B viruses undergo continuous antigenic changes, the live attenuated virus vaccines, like the licensed influenza virus subunit vaccines, will need to be updated frequently to be able to protect against the newly emerged epidemic antigenic variants of influenza A and B viruses. There are 15 antigenically distinct hemagglutinins (H1–H15) and nine antigenically distinct neuraminidases (N1–N9) in influenza A viruses that infect birds or mammals (98). Currently, two antigenic subtypes of influenza A virus (H1N1 and H3N2) co-circulate with influenza B virus in humans. A trivalent ca vaccine is therefore needed to protect against each of these three influenza viruses. The influenza A viruses possess a single-stranded, negative-sense RNA genome in eight segments that encode three polymerase proteins (98) (PB1, PB2, and PA); four membrane-associated proteins (the hemagglutinin [HA] glycoprotein, which mediates attachment and penetration; the neuraminidase [NA] protein, which mediates release of virus from the infected cell; the membrane protein [M], which lines the inner surface of the viral membrane and plays an essential role in virion structure and assembly, and the M2 protein, which forms an H1 ion channel that is required for release of the nucleocapsid following viral penetration of the host cell); a nucleocapsid protein (NP); a nonstructural protein (NS1) that functions as an interferon antagonist; a nuclear-export structural protein (NEP); and a newly described PB1-F2 protein (for influenza A viruses) that induces apoptosis in macrophages and lymphocytes (12). The genome of the influenza B viruses is organized similarly, but the NA gene contains a second open reading frame that encodes a second protein, NB, and the M gene does not encode an M2 protein (50). The NB protein of influenza B virus is thought to have a similar function as the M2 protein of influenza A virus (50). The segmented nature of the influenza virus genome permits the exchange of viral genes between two viruses co-infecting the same cell (Fig. 1). Such reassortment occurs between two influenza A viruses or two influenza B viruses but not between influenza A and B viruses. Protective immunity to influenza A viruses is mediated primarily by serum IgG and mucosal IgA antibodies directed against the HA and NA, making it necessary that these two antigens are derived from the newly emerged antigenic variant virus (98) (Fig. 1). Heterosubtypic immunity, that is, resistance to infection with an influenza A virus conferred by previ-

Antigenic variation in the hemagglutinin-neuraminidase protein of human parainfluenza type 3 virus.

Sixteen monoclonal antibodies directed to the hemagglutinin-neuraminidase (HN) protein of a 1957 isolate of parainfluenza type 3 virus (PIV3) were produced and used to examine antigenic variation in clinical strains. Analysis of hemagglutination-inhibition reactivity patterns of antigenic variants selected in vitro in the presence of monoclonal antibodies indicated that there were a minimum of six distinct epitopes detectable on the HN molecule. Competitive-binding assays indicated that these epitopes were located in two topologically nonoverlapping antigenic sites. An additional four epitopes were detected when 37 PIV3 clinical strains isolated over a period of 26 years in three geographic regions were tested for reactivity with the antibodies. Of the 10 unique epitopes defined by our monoclonal antibodies, 5 did not undergo detectable antigenic variation in any of the 37 strains examined. These results were expected since PIV3 viruses have been characterized as being antigenically monotypic. In contrast, antigenic variation was detected in the remaining five epitopes. This variation was not characterized by the accumulation of antigenic alterations with time (as for influenza A viruses), but appeared to represent genetic heterogeneity within the PIV3 population.

Expression of biologically active and antigenically authentic parainfluenza Type 3 virus hemagglutinin-neuraminidase glycoprotein by a recombinant baculovirus

The hemagglutinin-neuraminidase (HN) gene of human type 3 parainfluenza virus has been inserted into a baculovirus expression vector under the control of the polyhedrin promoter. HN protein produced in insect cells by the recombinant baculovirus appeared to be glycosylated, was transported to the cell surface, and was biologically active. All of the HN epitopes previously mapped functionally to a region(s) involved in neuraminidase and/or hemagglutination activities were conformationally unaltered on the recombinant protein. The HN produced in this system also induced a protective immune response in immunized cotton rats. From these studies we conclude that the HN expressed in insect cells represents a source of authentic HN glycoprotein suitable for structural analysis and immunization.

Antibody Responses to Bovine Parainfluenza Virus Type 3 (PIV3) Vaccination and Human PIV3 Infection in Young Infants

A phase 2 clinical trial was conducted to evaluate the antibody responses to bovine parainfluenza virus type 3 (bPIV3) vaccination in young infants. Three groups were tested as follows: placebo (n=66) and 10(5) (n=64) or 10(6) (n=62) TCID(50) of bPIV3. The vaccine or placebo was administered intranasally at ages 2, 4, 6, and 12-15 months, and serum specimens were collected at ages 2, 6, 7, 12-15, and 13-16 months. Serum hemagglutination inhibition (HI) and IgA antibody titers against bPIV3 and human PIV3 (hPIV3) were measured. The results indicate that antibody responses to bPIV3 vaccination are more likely to be detected by the bPIV3 IgA and HI assays than by the hPIV3 IgA and HI assays, that bPIV3-induced antibody response can be differentiated from hPIV3-induced antibody response most reliably by comparing bPIV3 and hPIV3 HI titers, and that bPIV3 vaccine prevents vaccine recipients from developing antibody profiles of hPIV3 primary infection.

ELECTROPORATION OF INFLUENZA VIRUS RIBONUCLEOPROTEIN COMPLEXES FOR RESCUE OF THE NUCLEOPROTEIN AND MATRIX GENES

Reverse genetics has been successfully used for the generation of recombinant influenza virus with altered biological properties. The standard method is based on DEAE-dextran transfection of in vitro reconstituted influenza virus ribonucleoprotein complex (RNP) into helper virus infected cells with subsequent selection of the recombinant viruses. Here we report the utilization of electroporation for reverse genetics of influenza virus as an improvement over the standard method. In a neuraminidase (NA) gene rescue system, we were able to demonstrate that electroporation of in vitro reconstituted NA RNP of influenza A/WSN/33 (H1N1) virus into WSN/HK virus infected cells allows the rescue of the transfectant WSN virus. The titer of transfectant virus obtained using electroporation is comparable to that generated using the DEAE-dextran transfection method. More significantly, the ratio of transfectant virus to helper virus is as much as 20-fold greater than that achieved using the DEAE-dextran system. We have also used electroporation to generate recombinant influenza virus carrying cDNA-derived matrix (M) gene or nucleoprotein (NP) gene of the WSN virus by using the temperature-sensitive (ts) mutants ts51 and ts56 as helper viruses. In the case of electroporation of M gene RNP, 88% of the viruses isolated after selection at 39 degrees C were transfectants. In contrast, the majority of viruses obtained using the DEAE-dextran transfection method were revertants of the helper virus. The NP-gene transfectant was only generated by the electroporation method. Our results suggest that electroporation of influenza virus RNP may be a useful method for generation of recombinant influenza viruses, especially in a system in which a ts mutant is used as helper virus.

Protection of cotton rats by immunization with the human parainfluenza virus type 3 fusion (F) glycoprotein expressed on the surface of insect cells infected with a recombinant baculovirus

The antigenicity, immunogenicity and efficacy of the human PIV3 fusion (F) glycoprotein expressed in insect cells by a baculovirus vector were studied. The results indicate that the PIV3 F glycoprotein expressed by a recombinant baculovirus is antigenically authentic as determined using a panel of PIV3 F specific monoclonal antibodies. Only a low level of antibody was stimulated by immunization of animals with infected cells, but the antibody appeared to be of high quality. Immunized animals were also moderately protected against PIV3 challenge. These results indicate that the baculovirus expression system is a reasonable source of authentic PIV3 F protein for use in a subunit vaccine.

Temperature sensitive mutants of influenza A virus generated by reverse genetics and clustered charged to alanine mutagenesis.

Temperature sensitive (ts) mutants of influenza A virus have the potential to serve as live attenuated (att) virus vaccines. Previously, ts mutants were isolated by chemical mutagenesis or arose spontaneously, and most likely contained point mutations in one or more genes. While sufficiently attenuated, even the most genetically stable of these viruses was found to revert to a more virulent form in a seronegative vaccinee. Recently developed technology, however, allows the introduction of engineered mutations into the genome of influenza A and B viruses, permitting the rational design of attenuated mutants with the potential for increased genetic stability. To accomplish this goal, we have introduced ts mutations into the PB2 gene of A/Los Angeles/2/87 (H3N2) and rescued the mutated genes into infectious viruses. We have used clustered charged to alanine mutagenesis (substitution of alanine for charged amino acid residues which are present in clusters) of the PB2 gene to generate novel ts mutants. Viruses containing such ts PB2 genes were attenuated in mice and ferrets. This approach has thus yielded several vaccine candidates with ts and attenuated characteristics in animal models. Combination of these mutations with each other or with other ts mutations may lead to a high level of genetic stability.

Interactions of a nonneutralizing IgM antibody and complement in parainfluenza virus neutralization

While many viruses activate the complement cascade directly, this is generally not a neutralizing event in the absence of antibody. We used a nonneutralizing IgM monoclonal antibody to parainfluenza virus type 3 (PIV3) hemagglutinin-neuraminidase (HN) to explore the role of antibody in complement-dependent neutralization of PIV3. Neither the antibody nor nonimmune guinea pig serum (GPS) neutralized PIV3 significantly, but a more than 100-fold reduction in titer was found when antibody and GPS were combined. Heat-inactivated GPS or GPS lacking either of two different complement proteins were all inactive with or without antibody. Specific repletion of the deficient sera with highly purified complement proteins restored neutralizing activity, indicating an absolute requirement for the classical pathway of complement activation and lytic terminal complement components, and viral lysis was confirmed by electron microscopy. The presence of antibody before complement activation was essential; later addition had no effect. Spontaneous complement activation by PIV3 occurred via the classical pathway in the absence of antibody. Addition of antibody did not alter the overall rate or extent of complement component C3 binding to PIV3 in these experiments. We conclude that certain nonneutralizing antibodies may support complement-dependent PIV3 neutralization by facilitating viral lysis. This process does not, however, involve enhanced activation through the C3 step. Lysis may require antibody-dependent localization of the membrane attack complex or reorganization of the viral envelope structures to facilitate attack complex insertion and lysis.

Neutralizing epitopes of human parainfluenza virus type 3 are conformational and cannot be imitated by synthetic peptides

The possibility that linear epitopes on the haemagglutinin-neuraminidase (HN) surface glycoprotein of human parainfluenza virus type 3 (PIV-3) might induce neutralizing antibodies after virus infection was investigated. Thirty-seven peptides, representing 64% of the extramembranous portion of the HN molecule of PIV-3, were synthesized. Their ability to bind to 14 neutralizing murine monoclonal antibodies (mAbs) specific for HN or 26 high-titre human serum samples were tested in a direct enzyme-linked immunosorbent assay (ELISA) and in an indirect competition ELISA. None of the synthetic peptides reacted with any of the mAbs or serum samples in the direct test and none of 11 synthetic peptides tested blocked mAbs from binding to HN in the competition ELISA. These findings suggest that synthetic peptides cannot be used to imitate the known neutralizing epitopes on the HN. Analyses of reduced and non-reduced HN in ELISA and immunoblot assays confirmed that protein folding and tertiary structure are essential for epitope formation in these neutralizing sites. However, some childrens sera analysed by immunoblotting contained antibodies to an uncharacterized linear epitope(s) not recognized by our panel of mAbs, raising the possibility that a neutralizing linear epitope does exist on the HN of PIV-3.