Measles Virus Hemagglutinin epitopes immunogenic in natural infection and vaccination are targeted by broad or genotype-specific neutralizing monoclonal antibodies.

Measles virus (MV) remains a leading cause of vaccine-preventable deaths in children. Protection against MV is associated with neutralizing antibodies that preferentially recognize the viral hemagglutinin (MV-H), and to a lesser extent, the fusion protein (MV-F). Although MV is serologically monotypic, 24 genotypes have been identified. Here we report three neutralization epitopes conserved in the more prevalent circulating MV genotypes, two located in the MV-H receptor binding site (RBS) (antigenic site III) and a third in MV-H/MV-F interphase (antigenic site Ia) which are essential for MV multiplication. In contrast, two MV-H neutralization epitopes, showed a genotype-specific neutralization escape due to a single amino acid change, that we mapped in the "noose" antigenic site, or an enhanced neutralization epitope (antigenic site IIa). The monoclonal antibody (mAb) neutralization potency correlated with its binding affinity and was mainly driven by kinetic dissociation rate (koff). We developed an immunoassay for mAb binding to MV-H in its native hetero-oligomeric structure with MV-F on the surface of a MV productive steady-state persistently infected (p.i.) human cell lines, and a competitive-binding assay with serum from individuals with past infection by different MV genotypes. Binding assays revealed that a broad neutralization epitope, in RBS antigenic site, a genotype specific neutralization epitopes, in noose and IIa sites, were immunogenic in natural infection and vaccination and may elicit long-lasting humoral immunity that might contribute to explain MV immunogenic stability. These results support the design of improved measles vaccines, broad-spectrum prophylactic or therapeutic antibodies and MV-used in oncolytic therapies.

Chimeric influenza-virus-like particles containing the porcine reproductive and respiratory syndrome virus GP5 protein and the influenza virus HA and M1 proteins.

Both porcine reproductive and respiratory syndrome and swine influenza are acute, highly contagious swine diseases. These diseases pose severe threats for the swine industry and cause heavy economic losses worldwide. In this study, we have developed a chimeric virus-like particle (VLP) vaccine candidate for porcine reproductive and respiratory syndrome virus (PRRSV) and H3N2 influenza virus and investigated its immunogenicity in mice. The HA and M1 proteins from the H3N2 influenza virus and the PRRSV GP5 protein fused to the cytoplasmic and transmembrane domains of the NA protein were both incorporated into the chimeric VLPs. Analysis of the immune responses showed that the chimeric VLPs elicited serum antibodies specific for both PRRSV GP5 and the H3N2 HA protein, and they stimulated cellular immune responses compared to the responses to equivalent amounts of inactivated viruses. Taken together, the results suggested that the chimeric VLP vaccine represents a potential strategy for the development of a safe and effective vaccine to control PRRSV and H3N2 influenza virus.

Plant-derived measles virus hemagglutinin protein induces neutralizing antibodies in mice.

Measles remains a significant problem in both the developed and developing world, and new measles vaccination strategies need to be developed. This paper examines the strategy of utilizing transgenic plants expressing a measles antigen for the development of an oral sub-unit measles vaccine. A 1.8 kb fragment encompassing the coding region of the measles virus hemagglutinin (H) protein was cloned into a plant expression cassette. Three different expression constructs were tested: pBinH (H gene alone), pBinH/KDEL (addition of a C-terminal endoplasmic reticulum-retention sequence SEKDEL) and pBinSP/H/KDEL (further addition of an authentic N-terminal plant signal peptide). The highest levels of recombinant H protein production were observed in plants transformed with pBinH/KDEL. Mice inoculated intraperitoneally with transgenic plant derived recombinant H protein produced serum anti-H protein antibodies that neutralized the measles virus (MV) in vitro. Mice gavaged with transgenic tobacco leaf extracts also developed serum H protein-specific antibodies with neutralizing activity against MV in vitro. These results indicate that the plant-derived measles H protein is immunogenic when administered orally and that, with further development, oral vaccination utilizing transgenic plants may become a viable approach to measles vaccine development.

Protective immunity against murine hepatitis virus (MHV) induced by intranasal or subcutaneous administration of hybrids of tobacco mosaic virus that carries an MHV epitope.

Hybrids of tobacco mosaic virus (TMV) were constructed with the use of fusion to the coat protein peptides of 10 or 15 amino acids, containing the 5B19 epitope from the spike protein of murine hepatitis virus (MHV) and giving rise to TMV-5B19 and TMV-5B19L, respectively. The TMV hybrids were propagated in tobacco plants, and the virus particles were purified. Immunogold labeling, with the use of the monoclonal MAb5B19 antibody, showed specific decoration of hybrid TMV particles, confirming the expression and display of the MHV epitope on the surface of the TMV. Mice were immunized with purified hybrid viruses after several regimens of immunization. Mice that received TMV-5B19L intranasally developed serum IgG and IgA specific for the 5B19 epitope and for the TMV coat protein. Hybrid TMV-5B19, administered by subcutaneous injections, elicited high titers of serum IgG that was specific for the 5B19 epitope and for coat protein, but IgA that was specific against 5B19 was not observed. Mice that were immunized with hybrid virus by subcutaneous or intranasal routes of administration survived challenge with a lethal dose (10 x LD50) of MHV strain JHM, whereas mice administered wild-type TMV died 10 d post challenge. Furthermore, there was a positive correlation between the dose of administered immunogen and protection against MHV infection. These studies show that TMV can be an effective vaccine delivery vehicle for parenteral and mucosal immunization and for protection from challenge with viral infection.

The roles of Fas/APO-1 (CD95) and TNF in antigen-induced programmed cell death in T cell receptor transgenic mice.

The possible involvement of Fas/APO-1 (CD95) and TNF in antigen-specific AICD of thymocytes and mature T cells has been investigated. Antigenic stimulation in vivo of influenza hemagglutinin (HA)-specific TCRtg mice was used to demonstrate that the kinetics of thymocyte and peripheral CD4+ T cell deletion are similar in mice with normal (+/+) or defective Fas (lpr/lpr) background, indicating that a Fas-independent pathway(s) is responsible for the deletion of activated T cells. TCRtg-+/+ or TCRtg-lpr/lpr mice injected with murine TNF-blocking MAb (TN3) showed rapid apoptosis of thymocytes after HA stimulation, indicating that death signaling through Fas and TNF receptors is not essential for HA-induced thymocyte deletion. CDC peripheral T cells in TCRtg-lpr/lpr mice did not undergo apoptosis following injection with HA and TN3, indicating that TNF-mediated apoptosis is involved in the deletion of mature T cells after antigenic stimulation. However, apoptosis still occurred in TCRtg-+/+ mice injected with TN3, indicating that both Fas- and TNF-mediated cell death can contribute to the deletion of activated peripheral T cells.

Liposomes that provide T-dependent help to weak antigens (T-independent antigens).

The design of an adjuvant for eliciting a thymus-dependent response to LPS, a well-defined thymus-independent antigen, is presented. Hybrid liposomes containing LPS and HA2 peptide from the hemagglutinin protein of influenza virus within the liposome bilayer were prepared (LPS/HA2 liposomes). The HA2 polypeptide contains epitopes recognized by T-helper lymphocytes and T-cytotoxic lymphocytes. Outbred mice immunized with LPS/HA2 liposomes produced anti-LPS-specific IgG responses. IgG subclass analysis indicated that IgG1, IgG2, and IgG3 antibodies were produced by these animals. LPS liposomes (liposomes without HA2) stimulated a T-independent response only. This was demonstrated by the detection of IgG3 but not IgG1 or IgG2 in serum of mice immunized with LPS liposomes. These results support the concept that the simultaneous incorporation into liposomes of a polypeptide with T-cell recognition sites along with a T-independent antigen can lead to the generation of cognate T-cell help for the T-independent antigen. The synthesis and characterization of a neo-lipopolysaccharide T-independent antigen for incorporation in hybrid HA2 liposomes are also presented. Findings are discussed relative to the liposome model used and implications for development of vaccines for use in humans.

Liposomes enhance the immunogenicity of reconstituted influenza virus A/PR/8 envelopes and the formation of protective antibody by influenza virus A/Sichuan/87 (H3N2) surface antigen.

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

Mechanism of immunity to influenza: maternal and passive neonatal protection following immunization of adult ferrets with a live vaccinia-influenza virus haemagglutinin recombinant but not with recombinants containing other influenza virus proteins.

Neonatal ferrets are protected against infection with influenza virus by milk-derived anti-influenza virus IgG after suckling on an immune mother. Live vaccines protect better than killed vaccines despite their stimulation of lower maternal haemagglutination-inhibiting antibody levels. This suggests that antibody to virus proteins other than the haemagglutinin may also be involved. To investigate this, adult ferrets were immunized intradermally with live vaccinia-influenza virus recombinants each expressing one of the 10 influenza virus polypeptides. Adult ferrets immunized with a recombinant expressing the H3 haemagglutinin were completely protected, and also passively protected their offspring, against a live challenge with clone 7a of the reassortant influenza virus A/Puerto Rico/8/34-A/England/939/69 (H3N2), immunity being mediated by IgG antibody. However, ferrets immunized similarly with recombinants expressing the H1 haemagglutinin, neuraminidase (N1 or N2), polymerases (PB1, PB2 or PAC), matrix protein (M1 or M2), nucleoprotein (NP) or non-structural proteins (NS1 or NS2) were completely susceptible to the influenza virus.

Induction and activity of class II-restricted, Lyt-2+ cytolytic T lymphocytes specific for the influenza H5 hemagglutinin.

In influenza A virus infections, CTL are a significant component of the host immune response which limits viral replication and promotes recovery. To examine the CTL response to the influenza virus A/Ty/Ont/7732/66[H5N9], particularly the H5 hemagglutinin, a long term CTL line was generated from spleen cells of A/Ty/Ont-immune Balb/c [H-2d] mice secondarily stimulated in vitro with A/Ty/Cal/Hurst-2/71[H5N2]. This CTL line was highly specific for influenza viruses of the H5 subtype. From this line, clones were isolated by limiting dilution and shown to be H5 hemagglutinin-specific based on recognition of an H5 vaccinia virus recombinant (H5 Vac). The clones exhibited the classical CTL surface phenotype Lyt-1-2+L3T4-; however, unlike the typically class I-restricted Lyt-2+ CTL, they were restricted in antigen recognition by class II (I-E) MHC molecules based on target cell recognition and antibody blocking of cytotoxicity. The clones recognized both infectious and non-infectious A/Ty/Ont presented by class II+ target cells. In adoptive transfer studies to assess the biologic role of the clones in vivo, these class II-restricted clones did not appear to alter mortality. However, these cells significantly reduced both morbidity and virus titers in the lungs of infected animals at 5 days post-infection. Thus, in the immune response to this virus, class II-restricted Lyt-2+ CTL specific for the H5 hemagglutinin were readily generated and their biologic role in vivo involved viral clearance.

Glycoproteins of human parainfluenza virus type 3: characterization and evaluation as a subunit vaccine.

The envelope glycoproteins of human parainfluenza type 3 virus were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reactivity with specific monoclonal antibodies. The molecular weight of the hemagglutinin-neuraminidase (HN) glycoprotein was found to be 72,000, and the fusion (F) glycoprotein appeared to consist of 74,000 (F0) or 56,000 (F1) species. Envelope glycoproteins were solubilized with octyl-glucoside and, after removal of the detergent by dialysis, were used for immunization of hamsters. Other animals were immunized with a formalin-inactivated preparation of whole virus. A single subcutaneous immunization with these antigen preparations induced a serum antibody response to the HN and F glycoproteins, as determined by plaque neutralization, hemagglutination inhibition, inhibition of virus-induced cell fusion, and immune precipitation tests. An IgG antibody response to both glycoproteins was also observed in bronchial washings. Animals immunized with the highest dose of envelope glycoproteins showed complete protection from challenge infection, whereas immunization with inactivated virus did not completely protect animals.

A synthetic decapeptide of influenza virus hemagglutinin elicits helper T cells with the same fine recognition specificities as occur in response to whole virus.

The immunogenicity of an isolated murine helper T cell determinant was studied. Mice were immunized with a synthetic peptide corresponding to amino acid residues 111-120 of the influenza PR8 hemagglutinin (HA) heavy chain, a region previously identified as a major target of the helper T cell response to the HA molecule in virus-primed BALB/c mice. Lymph node T cells from these mice were fused with BW 5147 cells to produce T hybrids for clonal analysis of their recognition specificities. Three T cell hybridoma clones, obtained from two different mice, responded to the immunizing peptide when presented by syngeneic antigen-presenting cells. All of these clones responded also to antigen provided as intact wild-type PR8 virus. The fine specificity of the peptide-induced T cell hybridomas, in response to a panel of mutant and variant influenza viruses, was indistinguishable from the fine specificities of T cells to the corresponding region of the HA1 chain of the HA molecule which had been generated by priming of mice with intact wild-type virus. These results suggest that an immunogenic determinant is contained within the 111-120 sequence that is able to elicit anti-influenza virus T cells with a similar repertoire to those elicited by immunization with whole virus.