Diphtheria antitoxin levels among children primed with a diphtheria and tetanus toxoids and acellular pertussis vaccine lot with a subpotent diphtheria toxoid component.

One lot of a nationally distributed diphtheria and tetanus toxoids and acellular pertussis (DTaP) vaccine was recalled in January 1999 because of a subpotent diphtheria toxoid component. To evaluate vaccine immunogenicity, children who had received the recalled lot for at least 2 of the 3 doses of their primary series were identified. Diphtheria antitoxin (DAT) levels were then prospectively assessed before and after dose 4 of (fully potent) DTaP vaccine. Of the 105 children evaluated, 84% had prevaccination DAT levels <0.10 IU/mL, which is the level generally accepted as protective. DAT levels rose a mean of 92-fold after dose 4; 100% of subjects had DAT levels >or=0.10 IU/mL, and 69% had DAT levels >or=1.0 IU/mL. These results indicate that diphtheria potency testing can identify vaccine that is less immunogenic when administered during the primary series. The booster response to dose 4, although reduced, was sufficient to confer adequate protection in the interval before receipt of the fifth dose of DTaP.

Validation of assays for use with combination vaccines.

A general methodology is presented for the validation of assays used for testing combination vaccines. The presentation is detailed and technical as our intention is to address challenges that we have encountered in the design and statistical analysis of assay validation studies. There are several noteworthy features which render the approach particularly useful in practice. It employs a statistical experimental design approach to the investigation of assay ruggedness with respect to manufacturing variability; it makes use of the assay variability results to determine the level of test-run replication necessary to achieve precision compatible with the product specifications; and, it provides a generic approach to assay validation. With combination vaccines, as with other pharmaceuticals, the analytical methods for release and stability must be validated early in the development programme Several things, though, distinguish this task with combination vaccines: (1) assays are typically pre-existing and often have been validated for use with an established sample matrix, e.g. a monovalent formulation; (2) sample matrices are complex and therefore more subject to manufacturing variability and more likely to cause assay interferences; and (3) the analytical workload is considerable due to the number of antigens. The methodology presented here was developed jointly by Merck Research Laboratories (West Point, PA) and Pasteur Mérieux Connaught, Inc. (Swiftwater, PA). Many of the issues discussed here have application outside of combination vaccines and are common features of all assay validations.

In previously immunized elderly adults inactivated influenza A (H1N1) virus vaccines induce poor antibody responses that are not enhanced by liposome adjuvant.

In a randomized, double-blinded study, 77 healthy elderly seropositive volunteers (95% of whom had received influenza vaccine within the prior 5 years) were immunized with either monovalent liposome-adjuvanted or control subvirion vaccine containing inactivated influenza A/Taiwan/1/86 (H1N1) virus. The experimental vaccine was well-tolerated but elicited serologic responses that were no different in frequency or magnitude from those induced by the control vaccine. Less than 20% of subjects in either group mounted a fourfold or greater rise in antibody titer. Sixty-three elderly subjects who had participated in the liposome vaccine trial were reimmunized 18 weeks later with licensed trivalent subvirion vaccine, and their serologic responses were compared with those of 26 young adults. Significant rises in hemagglutination inhibition (HAI) antibody titers to the A/Texas/36/91 (H1N1), A/Beijing/32/92 (H3N2) and B/Panama/45/90 components occurred in 10%, 76% and 56% of elderly vaccinees, respectively, compared to 92% (p < 0.0001), 100% (p < 0.005) and 88% (p < 0.005) of young vaccines, respectively. Age differences in seroresponse rates to the H1N1 subtype antigen were significant even when comparing young and old adults with identical prevaccination HAI antibody titers. These data confirm prior observations suggesting that previously immunized elderly persons have impaired serologic responses to influenza vaccines, particularly against recently circulating H1N1 subtype antigens. It remains unclear whether liposome-adjuvanted formulations would have an advantage over conventional influenza vaccines for routine annual reimmunization of targeted populations.

Cytotoxic T lymphocyte responses to a liposome-adjuvanted influenza A virus vaccine in the elderly.

This randomized double-blind study evaluated cytotoxic T lymphocyte (CTL) responses of elderly volunteers after parenteral immunization with either liposome-adjuvanted (n = 23) or control subvirion (n = 26) vaccine containing detergent-split influenza A/Taiwan/1/86 (H1N1) virus. Peripheral blood mononuclear cells obtained 0, 2, and 12 weeks after vaccination were stimulated in vitro with influenza A (H1N1) virus-infected autologous cells and then assayed for influenza virus-specific cytotoxicity using autologous virus-infected target cells. CTL responses to vaccination exhibited influenza A virus heterosubtypic cross-reactivity and were mediated primarily by CD8+ effector cells. Anti-influenza virus CTL activity was enhanced to a significantly greater extent by the liposome vaccine than by the control subvirion vaccine. It remains to be established whether the advantage of a liposomal formulation in terms of an improved CTL response is relevant to vaccine protective efficacy.

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.