Effect of priming on subsequent response to inactivated influenza vaccine.

Although shown to be a potent stimulator of serum antibody responses in animal models, the adjuvant immuno-stimulating complexes (ISCOMs) showed little adjuvant effect for inactivated influenza vaccines in a volunteer study. The result may be the non-comparability of the studies: animal studies were carried out chiefly in unprimed mice, while volunteers are mostly primed by previous infection and/or immunization. To test this, Balb/C mice were infected with influenza viruses or immunized with inactivated influenza vaccine, and subsequently given inactivated vaccine in saline or incorporated into ISCOMs. The serum in antibody responses was measured 1 month after immunization. The results confirm the adjuvant activity of ISCOM in unprimed mice, and show a marked reduction in adjuvant activity for primed mice. We argue that ISCOMs are important to prime the T cell response necessary for the serum antibody response to saline vaccine, but largely unnecessary where priming has been accomplished by prior exposure to influenza antigens. Further, the value of ISCOMs may lie in promoting antibody responses in unprimed subjects, and not in enhancing antibody titres.

Genetic differentiation between parapatric 'nearshore' and 'offshore' populations of the bottlenose dolphin.

The existence of nearshore and offshore populations of the bottlenose dolphin has been documented throughout its range. In several cases the two regional forms have been shown to be morphologically distinct, although there is considerable overlap for most characters. The populations off the eastern coast of North America have been the subject of a long-term programme of research on their distribution and movements. In this study, we compare mitochondrial and nuclear genetic markers between dolphins classified as either nearshore or offshore type. These putative populations were found to be distinct at both nuclear and mitochondrial genetic markers. Further, the level of variation among the nearshore dolphins was reduced compared with the offshore population. A broader geographical comparison suggests a shared lineage between offshore dolphins from the western North Atlantic and both offshore and nearshore dolphins from the eastern Atlantic. These results are consistent with local differentiation based on habitat or resource specialization in the western North Atlantic, and suggest differences in the character of the nearshore/offshore distinction in different parts of the world.

Induction of IL-2 and IFN-gamma in BALB/c mice immunised with subunit influenza A vaccine in combination with whole cell or acellular DTP vaccine.

Splenocytes from mice immunised with two doses of subunit influenza A/Beijing/353/89 vaccine mixed with whole cell DTP (wDTP), acellular DTP (aDTP) or PBS were collected 7 and 10 days after the second immunisation, and re-stimulated with subunit influenza vaccine or live virus in vitro. Interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) were assayed in supernates from these cultures by an ELISA procedure. Splenocytes from mice given subunit influenza vaccine in wDTP produced greater than two-fold and greater than five-fold responses of IL-2 and IFN-gamma, respectively, compared with splenocytes from mice immunised with subunit vaccine alone. In contrast, the response of splenocytes from mice immunised with subunit vaccine in saline or aDTP was similar, significantly less than for vaccine in wDTP (p < 0.01) and only slightly greater than for controls (p < 0.05). The production of IL-2 and IFN-gamma by these spleen cells was not significantly different on days 7 and 10 post-immunisation. Previous reports have shown that wDTP and aDTP enhance the serum antibody response of mice to influenza vaccine, but wDTP enhanced the response 100-fold greater than aDTP, and induced greater IgG2a and IgG2b subclass antibody responses; this last result indicates a cell-mediated immune response to vaccine. The present studies confirm these earlier findings; furthermore, as the IL-2 and IFN-gamma responses of splenocytes are associated with Th-1 subset T-lymphocyte response, the findings indicate a cytotoxic T-cell response to immunisation. The results indicate that influenza vaccine combined with wDTP induced a cell-mediated response in mice, which could confer a more solid immunity to challenge virus infection.

The nPCR detection of Chlamydia pneumoniae and Chlamydia trachomatis in children hospitalized for bronchiolitis.

Nasopharyngeal aspirates from children admitted with the diagnosis of bronchiolitis, were screened for Chlamydia pneumoniae, and C. trachomatis. The nested PCR was found to be more sensitive that amplified the DNA extracts of up to two elementary bodies of the test strains. Using this technique, C. pneumoniae was detected in two (1.3%), and C. trachomatis in 26 17.1%) of the 152 samples tested. This study indicated C. pneumoniae infections to be unusual in children with bronchiolitis.

A reverse transcriptase-PCR based assay for in-vitro antibiotic susceptibility testing of Chlamydia pneumoniae.

Infections caused by Chlamydia spp are an important cause of human disease, and the accuracy and reproducibility of antimicrobial susceptibility tests for these bacteria could have considerable clinical implications. We have developed a reverse transcriptase PCR (RT-PCR) based method to determine the antibiotic susceptibility of Chlamydia spp., and compared this with conventional tests using immunofluoresence (IF) staining. The MICs of antimicrobial agents for a test strain of Chlamydia pneumoniae were higher by RT-PCR as compared with IF staining, indicating the greater stringency of the former method. Using RT-PCR, doxycycline and tetracycline were the most active agents (MIC 1 mg/L), followed by erythromycin (1.6 mg/L), and ciprofloxacin (16 mg/L). Neither trimethoprim nor sulphamethoxazole (400 mg/L) inhibited growth as assessed by both techniques. The RT-PCR based method may thus represent an improved and less time consuming assay for in-vitro determination of the antibiotic susceptibility of Chlamydia spp.

The use of microbiology in the study of hygiene behaviour.

Faecal indicator bacteria have been used to measure levels of hygiene in a variety of settings. This paper describes a study in northern Botswana which used the isolation of faecal indicator bacteria in combination with other quantitative and qualitative techniques to gain information regarding hygiene behaviour. The microbiological samples included, samples from stored drinking water and water sources; eating plates; infant feeding bottles; dishcloths and the fingertips of carers and children. Water was usually clean at source but contaminated after storage. Presumptive faecal coliforms contaminated 31% of the eating plates, 29% of the dishcloths and 40% of the infant feeding bottles. Many of the presumptive faecal coliform isolates were not identified as Escherichia coli, indicating the need for further research into methodologies appropriate for isolating E. coli in tropical climates.

Immune response and protection against influenza A infection in mice immunised with subunit influenza A vaccine in combination with whole cell or acellular DTP vaccine.

Following the demonstration of the strong adjuvant effect of whole-cell DTP vaccine (wDTP) on the immune responses to influenza subunit vaccine, studies were undertaken to identify the component of wDTP responsible for the adjuvant effect, and to determine if acellular DTP (aDTP) vaccine was as effective since it is less reactogenic and likely to replace wDTP for primary or secondary immunisation. In addition, wDTP and aDTP were directly compared in a dose-response study. Experiments in mice indicated that the adjuvant effect of wDTP resided in the LPS component of B. pertussis, since purified LPS enhanced the IgG antibody response, the IgG subclass response and protection to the same level as wDTP. An adjuvant effect was detected using aDTP, but was statistically less pronounced than wDTP by a factor of some 100-fold. These results suggest that immunisation against influenza in infants and young children can be achieved combining small amounts of influenza antigen with wDTP or LPS, and to a lesser extent by combining vaccine with aDTP. However, these results were obtained in mice and should be confirmed in man since species vary considerably in response to adjuvant.

Immune response of mice to immunization with subunit influenza A vaccine in DTP vaccine.

Experiments were carried out to examine the initial feasibility of immunizing infants and children with inactivated influenza virus vaccine combined with diphtheria-tetanus-pertussis (DTP) vaccine. Groups of mice were immunized with saline vaccine or vaccine mixed with DTP: three doses of vaccine were given 3 weeks apart, and the antibody response and resistance to challenge infection were tested 3 weeks after the 1st and 3rd immunizations. The results showed that the antibody response and immunity to challenge virus infection were significantly greater for mice given vaccine in DTP than for mice given saline vaccine alone. Comparison of the response to graded doses of vaccine in saline or DTP indicated that vaccine in DTP was > 250-fold more effective in inducing serum antibody and protection than saline vaccine alone. The enhancing activity of DTP was significant for the alum component alone; however, most of the adjuvant effect was from the antigen components of the DTP vaccine. The results suggest that immunization against influenza in infants and young children could be achieved by combining small amounts of influenza antigen with DTP vaccines; however, the present results have been obtained in mice, and, since the responses to vaccines and adjuvants vary from species to species, the present results cannot be used to indicate similar results in human volunteers. The results indicate the potential value of an immunization procedure which should be tested in volunteers and which could provide a simple strategy for the immunization of at-risk infants and children against influenza.

Immunogenicity of influenza and HSV-1 mixed antigen ISCOMs in mice.

Immunostimulating complexes (ISCOMs) were prepared with mixtures of antigens from influenza A virus (A/PR/8/34 or A/Sichuan/2/87) and herpes simplex virus type 1 (HSV-1), and were characterised by enzyme linked immunosorbent assay (ELISA) and electron microscopy using double-labelling immunogold techniques employing monoclonal antibodies to influenza or HSV-1 glycoproteins. The immunogenicity of the mixed antigen ISCOMs was evaluated in mice, following administration by the subcutaneous route, by measuring the total and subclass IgG antibody responses. Protection of these animals against challenge with live influenza A/Sichuan virus or live HSV-1, was compared with that induced by immunization with aqueous mixed antigen preparations. It was found that relatively high humoral responses to both influenza and HSV antigens, and increased levels of protection to both influenza and HSV viruses were elicited in mice receiving the mixed antigen ISCOM preparation compared to those observed in animals receiving the mixed aqueous subunit preparation. The findings also indicate that antigens from more than one virus can be used in an ISCOM formulation to produce immunity and protection.

Comparative antibody responses and protection in mice immunised by oral or parenteral routes with influenza virus subunit antigens in aqueous form or incorporated into ISCOMs.

The total and subclass antibody responses of mice and protection of these animals against live influenza A/Sichuan/2/87 virus challenge infection were determined after immunisation with homologous A/Sichuan/87 aqueous or ISCOM-formulated surface glycoprotein subunit antigens administered by either the oral or intramuscular routes. The results show that the greatest systemic and local antibody responses were elicited in mice immunised with A/Sichuan ISCOMs by the intramuscular route; protection against homologous virus challenge was also effective in these animals, particularly after two doses of the vaccine. However, relatively high immune responses and protection were also elicited by the A/Sichuan/87 ISCOM vaccine administered orally. Immunisation of mice by the intramuscular route resulted in levels of serum IgG2a subclass antibody significantly greater than those induced by the same preparation given by the oral route, or by the aqueous A/Sichuan/87 subunit antigen preparation administered by either route. The findings indicate that the ISCOM delivery system can be used for immunisation by the oral route, although in mice, under the conditions used, this strategy compares unfavourably with the intramuscular route in terms of both local and systemic immune responses and protection against homologous challenge virus infection.

IgG subclass response and protection against challenge following immunisation of mice with various influenza A vaccines.

The serum total IgG and IgG subclass and nasal wash IgA and IgG antibody responses of mice to influenza virus A/Hong Kong/68 (H3N2) subunit preparations administered parenterally as a single dose, incorporated either in immune stimulatory compounds (ISCOMs) or liposomes with Freund's Complete Adjuvant, or as an aqueous material, as well as to live, infectious virus were measured by ELISA at 10 days and 3, 5, 7 and 22 weeks after immunisation. The protection of the upper and lower respiratory tracts provided by these preparations against homologous and heterologous challenge infection was assessed. Of the four variously-presented subunit preparations, influenza subunit ISCOMs induced relatively high and persisting levels of each of the different IgG subclasses, particularly IgG2a, throughout the study, and most nearly approached those observed after intranasal infection of mice with infectious virus. Furthermore, nasal wash IgA and IgG antibody levels, particularly at 5 or 7 weeks after immunisation, were also significantly greater in mice given the subunit ISCOM preparation than those induced by other subunit preparations with adjuvant or subunits given alone, and provided protection of both the upper and lower respiratory tracts against challenge as similar to that elicited by infectious virus.

Immunopotentiation of local and systemic humoral immune responses by ISCOMs, liposomes and FCA: role in protection against influenza A in mice.

The immunogenicity and protective efficacy of an influenza A subunit vaccine preparation administered to mice in an aqueous form, or presented as immunostimulatory complexes (ISCOMs), liposomes or with Freund's complete adjuvant (FCA), were assessed in comparative studies with live infectious virus. Both intranasal and parenteral routes of administration were assessed. An enzyme-linked immunosorbent assay (ELISA) was used to measure nasal wash and serum antibody responses in groups of unprimed mice, while protection was determined by the recovery of homologous influenza virus from mouse nasal washes and lung homogenates following challenge infection by the intranasal route. The results showed that parenteral administration of the influenza antigen preparations induced variable levels of both local and systemic antibodies at weeks 3, 7 and 22 postimmunization. Although the overall greatest levels of antibody and protection were elicited in mice following live virus infection, formulation of influenza surface haemagglutinin (HA) and neuraminidase (NA) proteins into ISCOMs elicited high and persistent antibody responses and provided relatively good protection of the upper and lower respiratory tracts of these animals. The results also show a relatively poor effect of the subunit antigen preparations in promoting humoral immune responses and protection irrespective of the nature of their presentation, when given by the intranasal route.

The subclass IgG responses of mice to influenza surface proteins formulated into liposomes.

Unprimed mice and mice primed by prior infection with an H1N1 subtype of influenza virus were used to assess the total and subclass IgG serum antibody responses to influenza virus A/Sichuan/2/87 (H3N2) surface haemagglutinin and neuraminidase proteins incorporated into four different formulations of liposomes. Only one of these liposome preparations, DSPC(B), induced greater total IgG, and subclass IgG1 and IgG2a antibody levels, in sera from both primed and unprimed mice than the aqueous A/Sichuan surface preparations alone administered at equivalent levels of haemagglutinin protein. The same DSPC(B) liposome formulation of A/Sichuan antigens was also the only preparation found to elicit levels of IgG2b and IgG3 subclass antibodies above baseline values in these animals.

Immune response of human volunteers and animals to vaccination with egg-grown influenza A (H1N1) virus is influenced by three amino acid substitutions in the haemagglutinin molecule.

Inactivated subunit vaccines were prepared from high-growth reassortants derived from two separate egg isolates from a single clinical specimen of influenza A (H1N1) virus. One of these reassortants, NIB-14, was antigenically indistinguishable from isolates made in tissue culture, while the other, NIB-17, was antigenically different and typical of egg isolates. The viruses differed by three amino acid residues in the haemagglutinin (HA) molecule and the anti-HA serological response induced was studied in animal models and human volunteers. In the volunteer groups both vaccines induced very high levels of circulating haemagglutination inhibition antibodies but with different serological specificities. Both NIB-14 and NIB-17 vaccines induced high levels of cross-reactive antibodies capable of reacting with both strains, but only NIB-14 vaccine induced significant levels of strain-specific antibodies capable of reacting exclusively with the homologous strain. Antisera containing only cross-reactive antibodies proved as capable of virus neutralization as antisera containing high levels of strain-specific antibodies. We extended the argument that epidemic strains are antigenically more closely related to tissue culture isolates and established that viruses which differ by only single amino acids at critical points in the HA structure can induce a significantly different immune response when used as inactivated vaccines.

The identification of informative parameters in the flow cytometric analysis of breast carcinoma.

DNA ploidy and the measurement of proliferation or S-phase fraction are both of prognostic significance in breast cancer, yet clinical use is minimal in the U.K. Immunohistochemistry is, however, used to aid diagnosis, so a panel of antibodies were analysed by flow cytometry to assess their predictive value for prognosis, tumour stage and grade. Of 10 parameters tested on 202 breast tumour samples, tumour cell proliferation and DNA ploidy were the two most informative; cytokeratin staining, natural killer and B-cell infiltration also proved to be of value but there was no prognostic value in measuring tumour infiltrating monocytes, helper/suppressor T-cell ratios, tumour cell reactivity with carcinoembryonic antigen or human milk fat globulin antibodies. For each of the informative parameters, scores numerically weighted towards a poorer prognosis were derived which when combined, correlated with tumour grade, stage and prognosis. Such data interpretation is objective, and can be transposed to other human tumours.

Non-random abnormalities of chromosomes 3, 6, and 8 associated with posterior uveal melanoma.

We present ten cases of posterior uveal melanoma which were karyotyped after short-term culture. One tumour had a normal chromosome complement. The remaining nine tumours were cytogenetically abnormal, with chromosomes 3, 6, 8, 11, and 13 most frequently involved. Abnormalities of chromosome 13 were seen in two cases, chromosome 11 in three cases, and chromosomes 3, 6, and 8 in five cases. Four tumours, all derived from the ciliary body, demonstrated monosomy 3 and i(8q), confirming the involvement of these aberrations with a subgroup of uveal melanomas arising from the ciliary body.

Detection of Chlamydia trachomatis by the polymerase chain reaction in young patients with acute epididymitis.

Specimens from 11 patients presenting with acute epididymitis were tested for the presence of Chlamydia trachomatis by an enzyme immunoassay (EIA), growth in McCoy cells and the polymerase chain reaction (PCR), and for other microorganisms by standard laboratory techniques. Chlamydia trachomatis urethral infection was detected in four patients by tissue culture, in three patients by EIA and in nine patients by PCR. These findings confirm the usually low detection rate of Chlamydia trachomatis by conventional tissue culture and EIA. Detection by PCR indicated both the diagnostic value of this technique and the importance of this organism in epididymitis.

Unique features of influenza viruses, and their implications.

Despite 50 years in intense research, influenza remains unique as the only truly pandemic viral infection of humans. In addition, although vaccines against this disease have been developed and used for the past four decades, they remain less effective than those used against other viral infections. Many of the reasons for this are now known, and depend on our understanding of the unique events that occur during virus replication. In particular, our understanding of virus variation following single infection, and the emergence of reassortant virus following double infection of cells, offers an explanation for many of the features of epidemic infection and forms a starting point for future scientific enquiry. Features of virus replication are discussed in this review, and the implications for the nature of epidemic infection and vaccine development are discussed.

The IgA and subclass IgG responses and protection in mice immunised with influenza antigens administered as ISCOMS, with FCA, ALH or as infectious virus.

Comparative studies on the local IgA, and circulating IgG subclass antibody responses of mice to A/Sichuan/2/87 (H3N2) influenza virus surface antigens administered with different carrier or delivery systems by the parenteral route, were carried out. The results obtained were compared with the responses observed following live influenza virus infection, and the protection afforded to these animals by these various preparations determined. Infection with live virus elicited early and high levels of protection against homologous virus challenge and this correlated with both local IgA and circulating IgG2a antibody levels. When incorporated into immunostimulating complexes (ISCOMS), A/Sichuan surface antigens promoted high levels of local IgA and circulating IgG1 antibody, and achieved a more rapid and more solid immunity against homologous virus challenge infection, than that elicited by the same surface antigens administered alone or together with Freund's complete adjuvant or alhydrogel.