Neutralizing antibody responses to Japanese encephalitis vaccine in children

Two shots of the current Japanese encephalitis (JE) vaccine were given to children and their immune responses to the Nakayama strain (the vaccine strain) and two wild strains (JaGAr-01 and E-50) of JE virus were examined by neutralizing (N) antibody titrations. Seventy vaccinees had no N antibody to JE virus before the first vaccination and were bled one month after the second vaccination. The N antibody responses to the JaGAr-01 and E-50 strains were found to be similar and to be less than that to the Nakayama strain after the second vaccination: the geometric mean titers (GMT) of N antibodies to the JaGAr-01 and E-50 strains (as logarithms) were 1.87 and 1.75, respectively, while the GMT to the Nakayama strain was 2.89. The seroconversion rates to the Nakayama, JaGAr-01 and E-50 strains were 70/70 (100%), 69/70 (99%) and 68/70 (97%), respectively, after the second vaccination. Twenty-seven of the 70 vacciness were also bled before the second vaccination. Most of them showed a considerably high N antibody response against the Nakayama strain and only one vaccinee failed to show seroconversion after the first vaccination. However, the antibody response to the E-50 strain appeared to be rather low and 9 of 25 vaccinees did not show any seroconversion. Similarly 3 of 25 failed to show any seroconversion against the JaGAr-01 strain. These results indicate that at the initial immunization two shots, at least, of the current JE vaccine are necessary to stimulate effective immune responses to wild strains of JE virus.     

Epidemiological and Ecological Studies of Japanese Encephalitis in Okinawa,Subtropical Area in Japan. II. Prevalence of Japanese Encephalitis Antibody in Residents in Okinawa,Miyako and Ishigaki Islands

During 1989 to 1990, human sera were collected by age groups in Okinawa (the northern, central and southern areas), Miyako and Ishigaki islands and examined for the neutralization (N) antibodies to two strains, Nakayama (vaccine strain) and C307 (Okinawan strain), of Japanese encephalitis (JE) virus. In Okinawa island, the N antibody positive rate to C307 was higher than that to Nakayama, while in Miyako and Ishigaki islands, the positive rate to Nakayama was higher than that to C307, suggesting that JE virus transmission rate was higher in Okinawa than in Miyako and Ishigaki islands. In Okinawa Prefecture, JE vaccine had not been administered to most of residents over 31 years of age at the time of serum collection. In residents over 31 years old, the positive rate to C307 was highest in the north of Okinawa (83.3%) and was lowest in Miyako (26.3%), with the second lowest in Ishigaki (33.3%). The distribution of N antibody titers to C307 gave hyperbolic patterns in the 0–5 age groups in Miyako and Ishigaki, and also in the 31–40, 41–50 age groups in Miyako and the 41–50 age group in Ishigaki, suggesting low rates of natural infection in these 4–5 decades in both islands. In residents of ages subjected to JE vaccine, a characteristic pattern was obtained, in which the curves to Nakayama shifted to higher titers than those to C307, suggesting that the first antigenic stimulation was caused by vaccine, not by natural infection of JE virus.     

Antigenic and Genetic Variations among Japanese Encephalitis Virus Strains Belonging to Genotype 1

Hyperimmune antisera against four Japanese encephalitis (JE) virus strains, ThCMAr4492 and ThCMAr6793 from Thailand and Nakayama and JaGAr01 from Japan, were used to analyze the antigenic relationships among 12 Thai strains belonging to genotype 1, and two Japanese strains and one Chinese strain belonging to genotype 3. The antiserum for ThCMAr6793 significantly neutralized nine of the 12 Thai strains, none of which was significantly neutralized by antisera for the Nakayama and JaGAr01 strains. The antiserum for ThCMAr4492 neutralized only its homologous strain; therefore, ThCMAr4492 was antigenically different from all other strains. Two Thai strains (Subin and KE-093/83) were significantly less neutralized by all four of the antisera tested. In the deduced amino-acid sequence of the E protein, the 12 Thai strains revealed 100 to 98.2% identity among them and 90.0 to 98.8% identity with the published strains, respectively. Among significant amino-acid substitutions, three residues at positions E-222, E-327 and E-366 were shared by all of the Thai strains, whereas residues at E-89, E-123, E-131, E-178, E-293, E-351 and E-373 seemed to be strain-specific. The amino acids at positions E-178, E-327, E-351, E-373 and E-366 are found either in the peptides with functional T-helper cell epitopes or in the ectodomain of the E protein of other flaviviruses. These amino acids may therefore be responsible for determining the antigenic heterogeneity of these strains.     

利用单克隆抗体对乙型脑炎病毒不同毒株抗原分析的研究

实验发现,应用动物免疫血清(多克隆抗体,PcAb)进行抗原分析时,我国分离到的乙型脑炎病毒SA14、P3、A2和高株的抗原性无明显差别,与从日本分离到的中山株有些差别,但仍被PcAb所中和;而用单克隆抗体(McAb)进行分析,情况就明显不同:SA14和P3株抗原性相似,高株和中山株不能被51-8McAb所中和,A2株则介于两者之间。A2和高顺生株的寡核苷酸指纹图谱分析表明,高株比A2株多两个斑点,但大多数斑点是一样的,证实了上述结果。实验还发现不同毒株的保护效果不同,A2株的免疫效果明显优于高株。     

Yellow fever/Japanese encephalitis chimeric viruses: construction and biological properties

A system has been developed for generating chimeric yellow fever/Japanese encephalitis (YF/JE) viruses from cDNA templates encoding the structural proteins prM and E of JE virus within the backbone of a molecular clone of the YF17D strain. Chimeric viruses incorporating the proteins of two JE strains, SA14-14-2 (human vaccine strain) and JE Nakayama (JE-N [virulent mouse brain-passaged strain]), were studied in cell culture and laboratory mice. The JE envelope protein (E) retained antigenic and biological properties when expressed with its prM protein together with the YF capsid; however, viable chimeric viruses incorporating the entire JE structural region (C-prM-E) could not be obtained. YF/JE(prM-E) chimeric viruses grew efficiently in cells of vertebrate or mosquito origin compared to the parental viruses. The YF/JE SA14-14-2 virus was unable to kill young adult mice by intracerebral challenge, even at doses of 10(6) PFU. In contrast, the YF/JE-N virus was neurovirulent, but the phenotype resembled parental YF virus rather than JE-N. Ten predicted amino acid differences distinguish the JE E proteins of the two chimeric viruses, therefore implicating one or more residues as virus-specific determinants of mouse neurovirulence in this chimeric system. This study indicates the feasibility of expressing protective antigens of JE virus in the context of a live, attenuated flavivirus vaccine strain (YF17D) and also establishes a genetic system for investigating the molecular basis for neurovirulence determinants encoded within the JE E protein.     

Antigenic variation among Sendai virus strains detected by monoclonal antibodies

Thirteen strains of Sendai virus isolated from various sources in the 1950's and after 1976 were compared for their reactivities with monoclonal antibodies prepared against the prototype strain MN of Sendai virus. Results revealed that while the 5 strains isolated in the 1950's reacted with all the monoclonal antibodies as the prototype strain did, the 2 strains isolated in 1976 and 1978 did not react with an F-specific monoclonal antibody, and the other 6 strains isolated after 1978 lacked reactivity with an HN-specific monoclonal antibody.     

Antigenic analysis of flaviviruses with monoclonal antibodies against Negishi virus

Twelve monoclonal antibodies against Negishi virus were obtained and characterized by hemagglutination inhibition (HI) and neutralization (NT) tests using five flaviviruses isolated in the pan-Pacific region. The reaction pattern of the antibodies showed that Negishi virus was most closely related to Langat virus, followed by 3-Arch, JE and Apoi viruses in that order. Hemagglutinating (HA) antigen of the virus had distinct HI relating sites which were Negishi virus specific, tick borne encephalitis (TBE) virus complex specific and flavivirus cross-reactive. Monoclonal anti-Negishi antibodies cross-reactive to Japanese encephalitis (JE) virus in the HI test had neutralizing activity to JE virus but no activity to homologous Negishi virus.     

Shift in S-layer protein expression responsible for antigenic variation in Campylobacter fetus.

Campylobacter fetus strains possess regular paracrystalline surface layers (S-layers) composed of high-molecular-weight proteins and can change the size and crystalline structure of the predominant protein expressed. Polyclonal antisera demonstrate antigenic cross-reactivity among these proteins but suggest differences in epitopes. Monoclonal antibodies to the 97-kDa S-layer protein of Campylobacter fetus subsp. fetus strain 82-40LP showed three different reactivities. Monoclonal antibody 1D1 recognized 97-kDa S-layer proteins from all C. fetus strains studied; reactivity of monoclonal antibody 6E4 was similar except for epitopes in S-layer proteins from reptile strains and strains with type B lipopolysaccharide. Monoclonal antibody 2E11 only recognized epitopes on S-layer proteins from strains with type A lipopolysaccharide regardless of size. In vitro shift from a 97-kDa S-layer protein to a 127-kDa S-layer protein resulted in different reactivity, indicating that size change was accompanied by antigenic variation. To examine in vivo variation, heifers were genetically challenged with Campylobacter fetus subsp. venerealis strains and the S-layer proteins from sequential isolates were characterized. Analysis with monoclonal antibodies showed that antigenic reactivities of the S-layer proteins were varied, indicating that these proteins represent a system for antigenic variation.     

Topographical analysis of antigenic determinants on envelope glycoprotein V3 (E) of Japanese encephalitis virus, using monoclonal antibodies.

Ten monoclonal antibodies directed against envelope glycoprotein V3 (E) of Japanese encephalitis virus were obtained. They were characterized by hemagglutination inhibition (HI), neutralization, and enzyme-linked immunosorbent assay and divided into four types: flavivirus-cross-reactive HI and non-neutralizing antibody (group 1), subgroup-specific HI and non-neutralizing antibody (group 2), low HI and neutralizing antibody (group 3), and non-HI and neutralizing antibody (groups 4 and 5, respectively). Competitive binding assays were performed to analyze the topography of antigenic determinants by enzyme-linked immunosorbent assay. The results of the competitive binding assay separated non-HI and neutralizing antibody into groups 4 and 5, respectively, and demonstrated the existence of at least five distinct antigenic determinants on V3. The site of group 1 was distinct from any other site. The sites of groups 2 and 3 seemed to be located close together. Our results suggest the following relationship between HI and neutralization: (i) The HI sites are separated from the neutralization sites, and (ii) there are two distinct HI sites, one of which is flavivirus cross-reactive, the other subgroup specific.     

A Similar Pattern of Interaction for Different Antibodies with a Major Antigenic Site of Foot-and-Mouth Disease Virus: Implications for Intratypic Antigenic Variation

The three-dimensional structures of the Fab fragment of a neutralizing antibody raised against a foot-and-mouth disease virus (FMDV) of serotype C₁, alone and complexed to an antigenic peptide representing the major antigenic site A (G-H loop of VP1), have been determined. As previously seen in a complex of the same antigen with another antibody which recognizes a different epitope within antigenic site A, the receptor recognition motif Arg-Gly-Asp and some residues from an adjacent helix participate directly in the interaction with the complementarity-determining regions of the antibody. Remarkably, the structures of the two antibodies become more similar upon binding the peptide, and both undergo considerable induced fit to accommodate the peptide with a similar array of interactions. Furthermore, the pattern of reactivities of five additional antibodies with versions of the antigenic peptide bearing amino acid replacements suggests a similar pattern of interaction of antibodies raised against widely different antigens of serotype C. The results reinforce the occurrence of a defined antigenic structure at this mobile, exposed antigenic site and imply that intratypic antigenic variation of FMDV of serotype C is due to subtle structural differences that affect antibody recognition while preserving a functional structure for the receptor binding site.     

Monoclonal antibodies against Chlamydia psittaci

Five monoclonal antibodies were prepared against Chlamydia (C.) psittaci strain Pigeon-1041 isolated from a feral pigeon in Sapporo. Reactions of these antibodies to chlamydiae were examined using five strains of C. psittaci and two strains of C. trachomatis in an enzyme-linked immunosorbent assay, microimmunofluorescent test and complement fixation test. The antibodies were divided into two groups: three genus-specific (A2, D2, and I21) and two strain-specific (F2 and H9) antibodies. The antigenic determinant site of A2 was KIO4 sensitive, but those of D2, F2, and H9 were not affected greatly by KIO4 treatment. Nine C. psittaci strains from feral pigeons and 16 strains from budgerigars were classified into three groups and four groups, respectively, by reaction patterns against the monoclonal antibodies.     

The dominant linear neutralizing antibody-binding site of glycoprotein gp86 of human cytomegalovirus is strain specific.

Bacterial fusion proteins, constructed from overlapping fragments of the open reading frame coding for gp86 of human cytomegalovirus (HCMV) strain AD169, were used to localize antigenic regions recognized by antibodies from human convalescent sera. A major domain for binding of conformation-independent antibodies was localized on fusion protein AP86, containing amino acids 15 to 142 of gp86. Human antibodies, affinity purified on AP86, neutralized infectious virus in tissue culture. In addition, a mouse monoclonal antibody (AP86-SA4), raised against AP86, also neutralized HCMV. AP86-SA4 was reactive with viral gp86 in immunoblot assays and showed a plasma membrane staining on intact HCMV-infected fibroblasts late in infection. After exonuclease III deletions of the viral gene, the binding site of neutralizing human as well as mouse antibodies was localized between amino acid residues 34 and 43. The domain has sequence variation between laboratory strains AD169 and Towne, and binding of the antibodies was strain specific. To our knowledge, this is the first characterization of a strain-specific neutralizing epitope on HCMV.     

Molecular and structural bases for the antigenicity of VP2 of infectious bursal disease virus

Infectious bursal disease virus (IBDV), a member of the family Birnaviridae, is responsible for a highly contagious and economically important disease causing immunosuppression in chickens. IBDV variants isolated in the United States exhibit antigenic drift affecting neutralizing epitopes in the capsid protein VP2. To understand antigenic determinants of the virus, we have used a reverse-genetics approach to introduce selected amino acid changes-individually or in combination-into the VP2 gene of the classical IBDV strain D78. We thus generated a total of 42 mutants with changes in 8 amino acids selected by sequence comparison and their locations on loops P(BC) and P(HI) at the tip of the VP2 spikes, as shown by the crystal structure of the virion. The antibody reactivities of the mutants generated were assessed using a panel of five monoclonal antibodies (MAbs). Our results show that a few amino acids of the projecting domain of VP2 control the reactivity pattern. Indeed, the binding of four out of the five MAbs analyzed here is affected by mutations in these loops. Furthermore, their importance is highlighted by the fact that some of the engineered mutants display identical reactivity patterns but have different growth phenotypes. Finally, this analysis shows that a new field strain isolated from a chicken flock in Belgium (Bel-IBDV) represents an IBDV variant with a hitherto unobserved antigenic profile, involving one change (P222S) in the P(BC) loop. Overall, our data provide important new insights for devising efficient vaccines that protect against circulating IBDV strains.     

Antigenic analysis of three strains of Mycoplasma arginini by two-dimensional immunoelectrophoresis.

The occurrence of species-specific and strain-specific antigens in three strains of Mycoplasma arginini (G-230, leonis and 23243) was studied by two-dimensional immunoelectrophoresis. Approximately 20 antigenic components could be detected in each strain. It was possible to analyze 6 to 7 major and distinct components from each strain by two techniques: "enhancement" where antigen to an additional strain is added to the first phase of the electrophoresis which increases the size of common peaks and "suppression" where antiserum to an additional strain is incorporated in the second phase whereby peak size of components to which both sera have antibody are decreased. A total of 10 distinct antigens were recognized. Electrophoretic mobilities relative to bovine albumin ranged from 0.2 to 1.08. Three components were common to all strains; two of these represented major amounts of material. Four components represented strain-specific components. Unique fast components were found both in strains 23243 and G-230. Three antigens were distributed into only two of the three strains. The electrophoretic mobilities of some common antigens were quite different between strains.     

Strain variation and nuclear association of Newcastle disease virus matrix protein.

Five monoclonal antibodies to the matrix (M) protein of Newcastle disease virus (NDV) Australia-Victoria (AV) strain were generated and characterized. In competitive antibody-binding assays, the antibodies fell into three discrete groups. The antigenic sites described by these antibody groups were designated M1, M2, and M3. Each antibody reacted with a panel of NDV strains in a manner unique to its group, confirming the grouping by competitive antibody binding. Only site M1 was found on all 12 of the strains tested and may be a "pan-NDV" epitope. A large portion of the M protein of strain AV was detected in the nuclei of infected cells by all five monoclonal antibodies. In addition, the antibodies only stained the nuclei of cells infected with NDV strains expressing M protein containing the corresponding antigenic site. These results confirm that the immunoreactivity in the nucleus is actually caused by the M protein and not by a cross-reacting host protein induced by viral infection.     

Antigenic relationship among the eight prototype and new serotype strains of Orientia tsutsugamushi revealed by monoclonal antibodies.

Orientia tsutsugamushi, the etiologic agent of tsutsugamushi disease, exhibits great antigenic variation. Three classical strains (Karp, Gilliam, and Kato) and new antigenic types from Thailand (TA686, TA678, TA716, TA763, and TH1817) have been used as prototype strains of O. tsutsugamushi in many studies. In this study, monoclonal antibodies to the five Thailand strains were produced, and their reactivity against prototype strains and newly identified isolates from Korea and Japan was tested. With a panel of these monoclonal antibodies, we could analyze the antigenic relationship among various strains of O. tsutsugamushi from Thailand, Japan, and Korea. Twelve strains of the O. tsutsugamushi tested showed various reactivities to monoclonal antibodies, and no distinct pattern of reactivity was found according to their location of isolation. Although the Boryong and Kuroki strains were similar in reactivities to most monoclonal antibodies, several monoclonal antibodies could differentiate the two strains. These results indicate that the immunofluorescence antibody test using monoclonal antibodies used in this study is valuable for analyzing the antigenic relationship and classification of O. tsutsugamushi.     

Neutralizing monoclonal antibodies against three serotypes of porcine rotavirus.

Using three serotypes (four strains) of cultivable porcine rotavirus as immunizing antigens, 10 neutralizing monoclonal antibodies were characterized. One VP4-specific monoclonal antibody directed against porcine rotavirus BEN-144 (serotype G4) neutralized human rotavirus strain ST-3 in addition to the homologous porcine virus. All nine VP7-specific monoclonal antibodies were highly specific for viruses of the same serotype as the immunizing rotavirus strain. One exception was the VP7-specific monoclonal antibody C3/1, which neutralized both serotype G3 and G5 rotaviruses. However, this monoclonal antibody did not neutralize the porcine rotavirus AT/76, also of serotype G3, nor mutants of SA-11 virus (serotype G3) which were selected with monoclonal antibody A10/N3 and are known to have mutations affecting the C antigenic region.     

A broadly neutralizing human monoclonal antibody that recognizes a conserved, novel epitope on the globular head of the influenza H1N1 virus hemagglutinin

The conserved influenza virus hemagglutinin (HA) stem domain elicits cross-reactive antibodies, but epitopes in the globular head typically elicit strain-specific responses because of the hypervariability of this region. We isolated human monoclonal antibody 5J8, which neutralized a broad spectrum of 20th century H1N1 viruses and the 2009 pandemic H1N1 virus. Fine mapping of the interaction unexpectedly revealed a novel epitope between the receptor-binding pocket and the Ca₂ antigenic site on HA. This antibody exposes a new mechanism underlying broad immunity against H1N1 influenza viruses and identifies a conserved epitope that might be incorporated into engineered H1 virus vaccines.