Michel Klein

Review of enterovirus 71 vaccines

Enterovirus 71 (EV71) and coxsackieviruses are the major causative agents of hand, foot, and mouth disease (HFMD) outbreaks worldwide and have a significant socioeconomic impact, particularly in Asia. Formalin-inactivated (FI) EV71 vaccines evaluated in human clinical trials in China, Taiwan, and Singapore were found to be safe and to elicit strong neutralizing antibody responses against EV71 currently circulating in Asia. The results from 3 different phase 3 clinical trials performed in young children (6-60 months) indicate that the efficacy of FI-EV71 vaccines is >90% against EV71-related HFMDs and >80% against EV71-associated serious diseases, but the vaccines did not protect against coxsackievirus A16 infections. Here we discuss the critical factors affecting EV71 vaccine product registration, including clinical epidemiology, antigenic shift issues in cross-protection and vaccine strain selection, standardized animal models for potency testing, and cost-effective manufacturing processes for potential incorporation of FI-EV71 vaccine into Expanded Programme on Immunization vaccines.

Cloning and expression of the Haemophilus influenzae transferrin receptor genes.

The genomic transferrin receptor genes (tbpA and tbpBu2003) from two strains of Haemophilus influenzae type b (Hib) and two strains of non‐typable H. influenzae (NTHi) have been cloned and sequenced. The deduced protein sequences of the H. influenzae tbpA genes were 95–100% conserved and those of the tbpB genes were 66–100% conserved. The tbpB gene from one strain of NTHi was found to encode a truncated Tbp2 protein. The tbpB genes from four additional NTHi strains were amplified by the polymerase chain reaction (PCR) utilizing primers derived from the conserved N‐terminal sequences of Tbp1 and Tbp2 and were found to encode full‐length proteins. Although several bacterial species express transferrin receptors, when the Tbp1 and Tbp2 sequences from different organisms were compared, there was only limited homology. Recombinant Tbp1 and Tbp2 proteins were expressed from Escherichia coli and antisera were raised to the purified proteins. There was significant antigenic conservation of both Tbp1 and Tbp2 amongst H. influenzae strains, as determined by Western blot analysis. In a passive model of bacteraemia, infant rats were protected from challenge with Hib after transfer of anti‐rTbp2 antiserum, but not after anti‐rTbp1 antiserum.

Production of EV71 vaccine candidates

Enterovirus 71 (EV71) is now recognized as an emerging neurotropic virus in Asia and with Coxsackie virus (CV) it is the other major causative agent of hand-foot-mouth diseases (HFMD). Effective medications and/or prophylactic vaccines against HFMD are urgently needed. From a scientific (the feasibility of bioprocess, immunological responses and potency in animal challenge model) and business development (cost of goods) points of view, we in this review address and discuss the pros and cons of different EV71 vaccine candidates that have been produced and evaluated in animal models. Epitope-based synthetic peptide vaccine candidates containing residues 211–225 of VP1 formulated with Freund’s adjuvant (CFA/IFA) elicited low EV71 virus neutralizing antibody responses, but were protective in the suckling mouse challenge model. Among recombinant EV71 subunits (rVP1, rVP2 and rVP3) expressed in E. coli, purified and formulated with CFA/IFA, only VP1 elicited mouse antibody responses with measurable EV71-specific virus neutralization titers. Immunization of mice with either a DNA plasmid containing VP1 gene or VP1 expressed in Salmonella typhimurium also generated neutralizing antibody responses and protected animals against a live EV71 challenge. Recombinant EV71 virus-like particles (rVLP) produced from baculovirus formulated either with CFA/IFA or alum elicited good virus neutralization titers in both mice and non-human primates, and were found to be protective in the suckling mouse EV71 challenge model. Synthetic peptides or recombinant EV71 subunit vaccines (rVP1 and rVLP) formulated in alum were found to be poorly immunogenic in rabbits. Only formalin-inactivated (FI) EV71 virions formulated in alum elicited cross-neutralizing antibodies against different EV71 genotypes in mice, rabbits and non-human primates but induced weak neutralizing responses against CAV16. From a regulatory, economic and market acceptability standpoint, FI-EV71 virion vaccines are the most promising candidates and are currently being evaluated in human clinical trials. We further describe and analyze some new bioprocesses technologies that have great potential applications in EV71 vaccine development. This review also demonstrates the opportunities and challenges that the Asian vaccine industry faces today.

Prospect and challenges for the development of multivalent vaccines against hand, foot and mouth diseases

Enterovirus 71 (EV71), an emerging neurotropic virus and coxsackieviruses (CV) are the major causative agents of hand, foot and mouth diseases (HFMD). These viruses have become a serious public health threat in the Asia Pacific region. Formalin-inactivated EV71 (FI-EV71) vaccines have been developed, evaluated in human clinical trials and were found to elicit full protection against EV71. Their failure to prevent CVA16 infections could compromise the acceptability of monovalent EV71 vaccines. Bivalent FI-EV71/FI-CVA16 vaccines have been found to elicit strong neutralizing antibody responses against both viruses in animal models but did not protect against CVA6 and CVA10 viral infections in cell culture neutralization assay. In this review, we discuss the critical bottlenecks in the development of multivalent HFMD vaccines, including the selection of vaccine strains, animal models to assess vaccine potency, the definition of end-points for efficacy trials, and the need for improved manufacturing processes to produce affordable vaccines.

Using serial observations to identify predictors of progression to aids in the Toronto Sexual Contact Study

The Toronto Sexual Contact Study comprises a cohort of 249 male sexual contacts of men with HIV disease which has been followed every 3 months for almost 5 years. On enrollment 143 were seropositive and 16 seroconverted during the follow-up period. By 31 December 1989, 41 of the 159 seropositive cohort members had developed AIDS. Using Cox relative risk regression models, we investigated the association of a number of laboratory and clinical variables and progression to AIDS. Fixed covariate models examined laboratory variables from the enrollment visit of cohort members, with time calculated from this date. In models assessing time dependent covariates, time was calculated from the estimated date of HIV infection. In the univariate models of either fixed or time dependent covariates, many variables were significantly associated with risk of progression to AIDS (T4 cell count, T4/T8 ratio, blastogenic responses to phytohemagglutinin, concanavalin A, and pokeweed mitogen, serum IgA, appearance of p24 antigen, and the development of oral hairy leukoplakia, thrush, or herpes zoster). Appearance of persistent generalized lymphadenopathy was not associated with increased risk of progression. In the multivariate model which evaluated fixed laboratory covariates, T4/T8 ratio, IgA level, and PHA response at enrollment were significantly associated with elevated risk.(ABSTRACT TRUNCATED AT 250 WORDS)

The IgA/IgM Receptor Expressed on a Murine B Cell Lymphoma Is Poly-Ig Receptor

T560, a mouse B lymphoma that originated in gut-associated lymphoid tissue, expresses receptors that bind dimeric IgA and IgM in a mutually inhibitory manner but have little affinity for monomeric IgA. Evidence presented in this paper indicates that the receptor is poly-Ig receptor (pIgR) known in humans and domestic cattle to bind both IgA and IgM. The evidence includes the demonstration that binding of IgM is J chain dependent, and that pIg-precipitated receptor has an appropriate Mr of 116–120 kDa and can be detected on immunoblots with specific rabbit anti-mouse pIgR. Overlapping RT-PCR performed using template mRNA from T560 cells and oligonucleotide primer pairs designed from the published sequence of mouse liver pIgR indicate that T560 cells express mRNA virtually identical with that of the epithelial cell pIgR throughout its external, transmembrane, and intracytoplasmic coding regions. Studies using mutant IgAs suggest that the Cα2 domain of dimeric IgA is not involved in high-affinity binding to the T560 pIgR. Inasmuch as this mouse B cell pIgR binds IgM better than IgA, it is similar to human pIgR and differs from rat, mouse, and rabbit epithelial cell pIgRs that bind IgA but not IgM. Possible explanations for this difference are discussed. All clones of T560 contain some cells that spontaneously secrete both IgG2a and IgA, but all of the IgA recoverable from the medium and from cell lysates is monomeric; it cannot be converted to secretory IgA by T560 cells.

Evaluation of the immunogenicity and protective efficacy of a candidate parainfluenza virus type 3 subunit vaccine in cotton rats

A parainfluenza virus type 3 (PIV3) subunit vaccine consisting of detergent-solubilized, affinity-purified haemagglutinin-neuraminidase (HN) and fusion (F) surface glycoproteins was tested in cotton rats for immunogenicity, short-term effects on virus-induced immunopathology and protective efficacy. Groups of animals were immunized twice, 4 weeks apart, with graded doses of vaccine administered either alone or with aluminium phosphate (AlPO4). The minimum immunogenic dose of vaccine was 0.1 microgram HN and F when the vaccine was given alone and 0.01 microgram when the vaccine was administered with AlPO4 adjuvant. Antibody responses in animals immunized with 1 microgram HN and F mixed with adjuvant were similar to those in control animals infected with live PIV3 intranasally. Pulmonary and nasal wash PIV3 titres generally were inversely correlated with serum antibody levels. Virus titres were significantly reduced in all groups of animals immunized with greater than or equal to 0.1 microgram HN and F compared with control animals immunized with vehicle only. Four days after virus challenge, there was no evidence of enhanced histopathology in lung sections from animals immunized with the candidate vaccine.

A monoclonal immunoglobulin G1 in which some molecules possess glycosylated light chains--I. Site of glycosylation.

Abstract A human monoclonal IgG1 κ protein (Hom) was found to possess two species of light chain with molecular weights of 23,000 and 28,000 respectively. The difference in mass was due to the presence of a carbohydrate prosthetic group on the 28,000 dalton species. The two species of light chain were separated by chromatography on CM-cellulose. Circular dichroism failed to detect any significant differences in conformation between the glycosylated and non-glycosylated forms and both species recombined with a heavy chain in an identical fashion as judged by difference spectroscopy. An anti-idiotypic antibody raised against the glycosylated Fab-fragment of IgGlHom was unable to distinguish between this species and the non-glycosylated Fab fragment, suggesting that the structure of the combining site is identical or very similar in both molecules. Amino-acid sequence analyses showed that both light chains had an identical sequence up to residue 41. Further studies on the glycosylated light chain, using o -iodosobenzoic acid fragments, localized the carbohydrate attachment site to asparagine 107, the penultimate residue of the J κ region which links V κ to C κ . The sequence around the attachment site was Asn-Arg-Thr which satisfies the requirements for an acceptor sequence. The non-glycosylated light chain was found to have the same sequence in this region indicating that the absence of glycosylation was not due to the lack of an acceptor sequence. A kinetic mechanism has been proposed to account for the incomplete glycosylation of the light chains of IgG1Hom in which there is a competition between the rate of folding of the nascent polypeptide and the rate of attachment of core sugars via the dolichol pyrophosphate pathway.

Non-covalent association of heavy and light chains of human immunoglobulin G: Studies using light chain labelled with a fluorescent probe☆

Abstract The fluorescent probe, 5-iodoacetamidofluorescein (5-IAF), was specifically attached to the COOH-terminal cysteine residue of the L chains of two monoclonal human IgKκ proteins. The induced circular dichroism and fluorescence properties of the bound 5-IAF probe were used to study changes in its microenvironment upon reassociation of autologous or heterologous L chains and their domains with Fd fragments. Recombination of 5-IAF-L with Fd at pH 5.4 was accompanied by a red-shifted visible difference spectrum, an increase in the magnitude of the induced optical activity and an enhancement of fluorescence. These results were compatible with the transfer of the 5-IAF moeity to a less polar and more asymmetric environment. Equilibrium and kinetic data obtained from difference spectroscopy and fluorescence studies indicated that 5-IAF-L bound with high affinity to Fd in a 1:1 ratio with a second-order rate constant of 1618 M −1 sec −1 at 25°C. Using the same approach, it was shown that the labelled constant region fragment (5-IAF-C κ ) bound to Fd with an association constant of 5 × 10 6 M −1 and a forward rate constant of 30 M −1 sec −1 . When 5-IAF-C κ was recombined with a preformed FdV κ complex, however, the intensity of the visible difference spectrum, the fluorescence emission, the binding affinity and the forward rate constant of the reaction, were significantly increased. In contrast, no spectroscopic changes were observed when V κ was recombined with a preformed Fd-5-IAF-C κ complex. These data suggest that: (1) the high affinity interaction between Fd and L results from the summation of relatively weak interactions between paired domains; (2) the binding of Vκ to Fd modulates the reactivity of Cγl toward Cκ, probably through conformation changes transmitted via V H -Cγl contacts; and (3) conversely, once 5-IAF-Cκ is bound to Fd, the addition of the complementary Vκ domains does not induce any detectable change in the vicinity of the fluorescent probe.

Effect of lipid modification on the physicochemical, structural, antigenic and immunoprotective properties of Haemophilus influenzae outer membrane protein P6

The outer membrane lipoprotein, P6 of Haemophilus influenzae was studied to determine the importance of the native palmitoyl moiety on its physicochemical and immunological properties. A recombinant P6 (rP6) molecule devoid of lipidation signal sequence was expressed in Escherichia coli and its properties were compared to those of the palmitylated protein purified from H. influenzae. The isoelectric point of rP6 was more acidic than that of the native protein and also exhibited less secondary structure than P6 as judged by circular dichroism. However, both forms of P6 induced identical P6-specific antibody titers in guinea pigs when Freunds adjuvant was used. These antisera reacted with a panel of overlapping P6 peptides in a comparable manner and in addition, rabbit antisera raised against the P6 peptides reacted equally well with P6 and rP6. Furthermore, all human convalescent sera tested exhibited similar anti-P6 and anti-rP6 antibody titers. However, rP6 was less immunogenic than P6 when administered either without adjuvant or in alum and when tested in competitive inhibition studies with anti-P6 antibodies, was a less effective inhibitor than native P6, suggesting a diminution in some of the antigenic activity of rP6. In spite of these differences, rP6 was capable of eliciting a protective antibody response against live H. influenzae type b challenge in a modified infant rat model of bacteremia. These findings demonstrate that the non-fatty acylated rP6 could possibily be substituted for native P6 in a vaccine against H. influenzae.