Toxicological evaluation of live attenuated, cold-adapted H5N1 vaccines in ferrets.

Live attenuated influenza vaccines (LAIV) have several attributes related to safety, immunogenicity, cross-protection against antigenic drift strains, high yield and needle-free administration that make them attractive candidates for control of pandemic influenza. H5N1 LAIV vaccine candidates are attenuated in ferrets, chickens and mice. These vaccine candidates were further characterized in the ferret model to evaluate their toxicity at doses comparable to seasonal LAIV and at doses up to 100-fold higher. The results demonstrated that H5N1 LAIV, even when administered at high doses, is restricted in replication in the lower respiratory tract of ferrets. However, intranasal administration of 0.5 mL can result in deposition of H5N1 LAIV in the ferret lung, where it induces a pulmonary inflammatory response in the absence of significant local replication of the vaccine virus. Thus, smaller vaccine dose volumes should be considered for evaluation of LAIV in animal models.

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.

Monoclonal antibody-resistant mutants selected with a respiratory syncytial virus-neutralizing human antibody fab fragment (Fab 19) define a unique epitope on the fusion (F) glycoprotein.

A recombinant human antibody fragment, designated RSV Fab 19, efficiently neutralizes respiratory syncytial virus (RSV). Here we report the results of our sequence analysis of antibody escape mutants that identified F glycoprotein amino acids critical for binding of human or murine RSV F-neutralizing antibodies.

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.

Human cytomegalovirus neutralizing antibody-resistant phenotype is associated with reduced expression of glycoprotein H.

We have characterized a neutralizing antibody-resistant mutant human cytomegalovirus (HCMV) obtained from a patient treated with a human monoclonal antiglycoprotein H (gH; unique long region 75) antibody. This virus exhibited resistance to several different neutralizing anti-gH murine monoclonal antibodies (MAbs), as well as to a polyvalent anti-gH serum. The resistant phenotype was unstable and could be maintained only by passage of plaque-purified virus under neutralizing MAb selection. In the absence of a MAb, the resistant phenotype reverted to a neutralizing antibody-sensitive phenotype within one passage. The predicted amino acid sequences of gH from the MAb-resistant and -susceptible parent viruses were identical. Biochemical analysis of the MAb-resistant and -susceptible parent viruses revealed a marked decrease of gH expression in the envelope of the MAb-resistant virus. Furthermore, propagation of the virus in various MAb concentrations resulted in the production of extracellular virions with various levels of resistance to the neutralizing activity of the MAb. These results suggest a mechanism for the generation of neutralizing antibody-resistant viruses which could evade host-derived antiviral antibody responses. In addition, our findings indicate that the stoichiometry of gH in the envelope of infectious HCMV virions is not rigidly fixed and therefore offer a simple explanation for production of phenotypic variants of HCMV through an assembly process in which the content of gH in the envelope of progeny virions varies randomly.

Neutralizing monoclonal antibodies that distinguish three antigenic sites on human cytomegalovirus glycoprotein H have conformationally distinct binding sites.

Seven neutralizing murine monoclonal antibodies specific for the glycoprotein H of human cytomegalovirus were produced and used to construct a topological map of two nonoverlapping antigenic sites that are bridged by a third antigenic site. Neutralization assays with 15 laboratory or clinical human cytomegalovirus strains indicated that the monoclonal antibodies recognize three antigenically variable and three conserved epitopes within the three antigenic sites. The variable-domain genes encoding monoclonal antibodies representing each of the three antigenic sites were cloned and sequenced, and molecular models of their binding sites were generated. Conformational differences in the antibody-binding sites suggested a structural basis for experimentally observed differences in gH epitope recognition.

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.

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 children's 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.

Antibody responses of humans and nonhuman primates to individual antigenic sites of the hemagglutinin-neuraminidase and fusion glycoproteins after primary infection or reinfection with parainfluenza type 3 virus.

An unusual feature of human parainfluenza virus type 3 (PIV3) is ita ability to cause reinfection with high efficiency. The antibody responses of 45 humans and 9 rhesus monkeys to primary infection or subsequent reinfection with PIV3 were examined to identify deficiencies in host immunologic responses that might contribute to the ability of the virus to cause reinfection with high frequency. Antibody responses in serum were tested by using neutralization and hemagglutination inhibition (HI) assays and a monoclonal antibody blocking immunoassay able to detect antibodies to epitopes within six antigenic sites on the PIV3 hemagglutinin-neuraminidase (HN) glycoprotein and eight antigenic sites on the fusion (F) protein. Primary infection of seronegative infants or children with PIV3 stimulated strong and rather uniform HI and neutralizing antibody responses. More than 90% of the individuals developed antibodies to four of the six HN antigenic sites (including three of the four neutralization sites), but the responses to F antigenic sites were of lesser magnitude and varied considerably from person to person. Young infants who possessed maternally derived antibodies in their sera developed lower levels and less frequent HI, neutralizing, and antigenic site-specific responses to the HN and F glycoproteins than did seronegative infants and children. In contrast, children reinfected with PIV3 developed even higher HI and neutralizing antibody responses than those observed during primary infection. Reinfection broadened the HN and F antigenic site-specific responses, but the latter remained relatively restricted. Adults possessed lower levels of HI, neutralizing, and antigenic site-specific antibodies in their sera than did children who had been reinfected, suggesting that these antibodies decay with time. Rhesus monkeys developed more vigorous primary and secondary antibody responses than did humans, but even in these highly responsive animals, response to the F glycoprotein was relatively restricted following primary infection. Bovine PIV3 induced a broader response to human PIV3 in monkeys than was anticipated on the basis of their known relatedness as defined by using monoclonal antibodies to human PIV3. These observations suggest that the restricted antibody responses to multiple antigenic sites on the F glycoprotein in young seronegative infants and children and the decreased responses to both the F and HN glycoproteins in young infants and children with maternally derived antibodies may play a role in the susceptibility of human infants and young children to reinfection with PIV3.

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.

Attenuation of bovine parainfluenza virus type 3 in nonhuman primates and its ability to confer immunity to human parainfluenza virus type 3 challenge.

Bovine parainfluenza virus type 3 (PIV-3) was evaluated as a candidate live-virus vaccine to protect against infection with human PIV-3. The level of replication of bovine and human PIV-3 and the efficacy of immunization with bovine PIV-3 in protecting against subsequent challenge with human PIV-3 was evaluated in nonhuman primates. The duration and magnitude of replication of human and bovine PIV-3 in the upper and lower respiratory tracts of New World monkeys was similar, and animals infected with bovine PIV-3 developed resistance to challenge with human PIV-3. The replication of two bovine strains of PIV-3 was restricted 100- to 1000-fold in Old World primates but was sufficient to induce high levels of neutralizing antibody to human PIV-3. The combined properties of restricted replication and induction of a protective immune response to human PIV-3 in nonhuman primates make bovine PIV-3 a promising candidate for a live-virus vaccine to protect humans against disease caused by PIV-3.

Nucleotide and deduced amino acid sequence of hemagglutinin-neuraminidase genes of human type 3 parainfluenza viruses isolated from 1957 to 1983.

We have sequenced the coding and noncoding regions of the hemagglutinin-neuraminidase (HN) genes of six clinical strains of human type 3 parainfluenza virus (PIV3) isolated between 1973 and 1983, and compared them to the prototype 1957 strain. Sequence variability does not result from the accumulation of mutations over time, but represents genetic heterogeneity in HN genes within the PIV3 population. Most of the nucleotide diversity occurs in the 5' noncoding sequences, exclusive of regions supplying transcriptional and translational control elements. Although the overall amino acid homology among HN proteins is very high, most variability is concentrated in domains at the carboxyl and amino terminus. This uneven distribution of amino acid diversity may reflect both functional and structural constraints on different HN domains and the epidemiologic features of PIV3 infection.

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.

Antigenic and structural properties of the hemagglutinin-neuraminidase glycoprotein of human parainfluenza virus type 3: sequence analysis of variants selected with monoclonal antibodies which inhibit infectivity, hemagglutination, and neuraminidase activities.

The hemagglutinin-neuraminidase (HN) gene sequence was determined for 16 antigenic variants of human parainfluenza virus type 3 (PIV3). The variants were selected by using monoclonal antibodies (MAbs) to the HN protein which inhibit neuraminidase, hemagglutination, or both activities. Each variant had a single-point mutation in the HN gene, coding for a single amino acid substitution in the HN protein. Operational and topographic maps of the HN protein correlated well with the relative positions of the substitutions. There was little correlation between the cross-reactivity of a MAb with the bovine PIV3 HN and the amount of amino acid homology between the human and bovine PIV3 HN proteins in the regions of the epitopes, suggesting that many of the epitopes are conformational in nature. Computer-assisted analysis of the HN protein predicted a secondary structure composed primarily of hydrophobic beta sheets interconnected by random hydrophilic coil structures. The HN epitopes were located in predicted coil regions. Epitopes recognized by MAbs which inhibit neuraminidase activity of the virus were located in a region which appears to be structurally conserved among several paramyxovirus HN proteins and which may represent the sialic cid-binding site of the HN molecule.

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.