不同佐剂条件下Aβ多肽B细胞表位疫苗诱导产生抗体的免疫反应特性分析

目的:研究阿尔茨海默病β淀粉样肽(Aβ)B细胞表位疫苗2Aβ1-15-PADRE(Aβ-T)诱导产生抗体的免疫反应特性,并探讨不同佐剂对该疫苗免疫反应效果的影响。方法:合成了含2个Aβ42的 B细胞表位—Aβ1-15及1个辅助T细胞表位—PADRE的多肽2Aβ1-15-PADRE。采用Al(OH)₃佐剂,弗氏佐剂,Abisco佐剂,MF59佐剂分别与多肽疫苗联合免疫小鼠,并另设3个对照组:无佐剂多肽免疫组(Mock),PBS免疫组(PBS),未免疫组(Native)。结果:5组多肽免疫组小鼠均产生了针对Aβ的特异性抗体,无佐剂多肽免疫组的IgG抗体滴度最低,Al(OH)₃佐剂组,MF59佐剂组,Abisco佐剂组小鼠IgG抗体滴度较高,弗氏佐剂组IgG抗体滴度最高。斑点杂交实验结果显示5组小鼠免疫后血清与Aβ42单体反应较弱,与寡聚体反应最明显,与纤维状Aβ42几乎不反应。结论:4种佐剂均能提高多肽疫苗的免疫反应,产生高水平抗Aβ的特异性抗体。5组免疫小鼠产生的抗体均与Aβ寡聚体反应较强,与纤维状Aβ42反应较弱,表明该多肽疫苗具有良好的应用前景。     

Saponin Adjuvants. IV. Evaluation of The Adjuvant Quil a in the Vaccination of Cattle Against Foot-And-Mouth Disease

The saponin adjuvant Quil A has been investigated in the vaccination of cattle against foot-and-mouth disease. Using a Frenkel type vaccine a dose-response relationship has been established between Quil A and neutralizing antibody titres. Ten ml of vaccine was combined with 0, 50, 200, 800, and 3200 µg of Quil A. The combinations were each injected into 4 animals. The local reaction on the site of injection produced by injection of the vaccine alone and in combination with different doses of Quil A has been estimated. On this basis a therapeutical dose at 1 mg of Quil A has been estimated to combine maximum adjuvant effect with a minimum of adverse reactions. This dose has been tested in the vaccination of cattle with FMD vaccines derived from BHK suspension cell virus of type O and A respectively. The vaccines were tested in 10 ml and 5 ml doses with or without Quil A, and each in 4 animals. It is concluded that Quil A is a valuable adjuvant for use in the induction of neutralizing antibodies against foot-and-mouth disease in cattle.     

Immune response in cattle vaccinated against rabies

In order to determine the best type of rabies vaccine to use as a booster, 78 serological samples from singly vaccinated cattle were analyzed by counterimmunoelectrophoresis technique. The animals were divided into several groups, received the first vaccine dose with modified live virus vaccine (ERA strain) and were revaccinated with inactivated virus or modified live virus vaccines. Boosters were given at 2, 4, 8, 12 and 16 weeks following first vaccination. Results showed high titres in the cases of booster with inactivated vaccine. In all cases, however, detectable antibody titres declined quickly.     

The response of cattle vaccinated with hypodermin A to a natural infestation of Hypoderma bovis and Hypoderma lineatum.

In this study, we have determined whether immunization with hypodermin A (HA), associated with various adjuvants, could provide protective immunity for calves when challenged with a natural hypoderma infestation. Groups of naive calves were vaccinated with HA antigen alone or with adjuvants [Freund's incomplete adjuvant (FIA) or alumina phosphate (AP)]. Subcutaneous injection with HA antigen with or without adjuvant did not significantly protect calves against a natural hypodermosis infestation. The humoral response during the infestation period was evaluated by ELISA. A significant earlier and greater response was induced in groups vaccinated with HA alone and HA combined with FIA. These results indicate that HA, in this vaccination protocol, induces a very incomplete protection in calves exposed to a natural infestation.     

Immunogenicity and protective efficacy of solubilized merozoite-enriched Theileria sergenti immunogens. II: Protection against natural exposure under field conditions.

A Theileria sergenti soluble merozoite preparation containing the 29, 34, 35 and 105 KD as the immunodominant polypeptides, was evaluated for efficacy, safety and protectivity in Holstein calves against virulent field tick challenge. The soluble antigens (100 mg/dose) were fortified with either complete or incomplete Freund's adjuvant. Twenty naive calves, aged one month, were subcutaneously inoculated with the preparation and a booster dose was administered 4 weeks later. Twenty additional calves served as controls. Five weeks after the booster dose, vaccinates and uninoculated controls were moved to a pasture, a heavily tick infested area in Cheju-do, Korea. The vaccinates showed negligible change in hematocrit and total RBC count whereas control animals showed significant (p < 0.05) hematological changes and associated anemia. Only 30% of vaccinates required chemotherapy after the experiment was terminated. All control animals required chemotherapy and 25% received blood transfusion. The highest percent parasitized erythrocytes in vaccinated cattle was 0.4% as compared with 3.6% among controls during the month of July. A significant difference (p < 0.05) was observed in the rate of body weight increase. Significant differences were also noted in serum albumin, aspartate aminotransferase, total protein and bilirubin. Significantly more vaccinated cattle maintained normal ranges of hematological and biochemical values as compared with the control group. It is suggested that soluble merozoite T. sergenti antigens may be potential vaccine candidates for developing a genetic vaccine in Korea.     

Vaccines against blowfly strike: the effect of adjuvant type on vaccine effectiveness.

Vaccination of sheep with a partially purified extract of Lucilia cuprina larvae in some cases resulted in marked reduction of growth in larvae which fed on the sheep. Twelve adjuvants were assessed, in vitro and in vivo, to determine which induced the largest inhibitory effect on larval growth. The Freund's complete adjuvant and Quil A groups produced ELISA antibody levels significantly higher (P less than 0.05) than other groups. Seven adjuvants mediated an immune response which caused significant inhibition of larval growth (P less than 0.05). When the sheep were assessed by in vivo larval culture, only larvae feeding on sheep vaccinated with the antigen presented in Freund's complete adjuvant or dextran sulphate or a dextran sulphate/Freund's incomplete adjuvant mixture weighed significantly less (P less than 0.05) than larvae feeding on control sheep. The effect on larvae was monitored in vitro for 70 days after vaccination, by which time significant reduction in larval weight was no longer observed. The loss of larval growth inhibition was not associated with a corresponding reduction in overall antibody levels.     

Stable biocompatible adjuvants--a new type of adjuvant based on solid lipid nanoparticles: a study on cytotoxicity,compatibility and efficacy in chicken

A new type of adjuvant was tested for its ability to initiate antibody production in chickens, and its cellular and tissue compatibility were assessed. The stable biocompatible adjuvants tested are based on surface-modified solid lipid nanoparticles (SLNs), made from paraffin or biodegradable glycerides, and are simply admixed to the antigens before administration. The tissue-damaging potency of four formulations of the new adjuvants (H1, H2, H3 and H4) were first tested in vitro by using human foreskin fibroblasts and RAW 264.7 macrophages. The adjuvants were well tolerated by both cell types. Immunisation studies in chickens were performed by using a Mycoplasma bovis antigen and mouse immunoglobulin G (IgG). The resulting antibodies were non-invasively extracted from egg yolk. The use of the various adjuvant formulations resulted in a significant production of specific antibodies after the first and second booster immunisations. Freund's complete adjuvant (FCA), considered until now to be the "gold standard" among the adjuvants, revealed the highest antibody titre against mouse IgG. SLNs with a particle size of more than 100 nm exhibited a clear adjuvant activity, whereas SLNs with a particle size below 100 nm, in various concentrations, revealed a lower adjuvant activity. Immunisation of chickens with the mouse IgG alone, dissolved in phosphate-buffered saline, resulted in a slow antibody titre development. At the end of the experiment, the chickens were examined for vaccination-associated tissue damage. In contrast to FCA, the SLN formulations caused only minor tissue irritation at the injection sites. In conclusion, SLNs seem to be a promising alternative to FCA for antibody production in chickens, and potentially in other animals.     

Preparation and Effect of Different Adjuvants on the Immunogenic Activity of Mycobacterial Ribosomal Fraction

Several emulsified and two nonemulsified incomplete adjuvants were examined for their adjuvant activity by use of mycobacterial ribosomal fractions as a substrate. A good adjuvant is defined as one which produces a high immunological response with the ribosomal fraction in mice to infection with virulent tubercle bacilli. Freund's incomplete adjuvant, consisting of Aquaphor and heavy mineral oil, and Arlacel A plus hexadecane were the best adjuvants tested. Aquaphor plus light mineral oil and Arlacel A plus 7-n-hexyloctadecane were not quite as effective. Peanut oil was not satisfactory when emulsified with either Aquaphor or Arlacel A. A moderate degree of immunity was produced in mice vaccinated with ribosomal fraction mixed with aluminum hydroxide gel. Sodium alginate mixed with ribosomal fraction produced a low degree of immunity only with the highest vaccinating dose. It was found that the effectiveness of the emulsified type of adjuvant depended upon the method of preparation. Careful standardization of technique to produce uniform and complete emulsification was essential for maximal adjuvant activity using minimal vaccinating doses. A rapid and practical method of preparing emulsified adjuvants is given. The mode of action of incomplete adjuvants as employed in these experiments is discussed, and it is thought that they acted primarily by protecting the ribosomes from being inactivated by host ribonuclease before they were engulfed by the macrophages.     

Rotavirus vaccine administered parenterally induces protective immunity.

We performed experiments to determine whether parenteral immunization with SA11 rotavirus can induce active protective immunity in a rabbit model of rotavirus infection. After one or two intramuscular injections of 1 ml of live or formalin-inactivated SA11 virus, we evaluated the mucosal and serologic immune response and protection from challenge with a high dose of live, virulent rabbit (Ala) rotavirus. Inactivated SA11 virus preparations, evaluated by enzyme-linked immunosorbent assay (ELISA) with a panel of VP4- and VP7-specific neutralizing and nonneutralizing monoclonal antibodies, did not show a loss of epitopes from the inactivation procedure compared with live virus. Administration of two doses of vaccine, one at zero days postvaccination (DPV) and a booster shot at 49 DPV, followed by challenge at 71 DPV with 3.5 x 10(5) PFU of Ala virus resulted in protection from challenge. None of the two-dose virus-vaccinated rabbits shed virus after challenge, while virus shedding was detected in all control rabbits (P = 0.001, Fisher's exact two-tailed test). Differences in total serum immunoglobulin (Ig) antirotavirus ELISA titers (P < 0.05, Wilcoxon's rank sum test) were observed between groups vaccinated with virus in aluminum phosphate or Freund's adjuvant but not between groups vaccinated with live or inactivated virus in either adjuvant. All rabbits given two doses of vaccine had detectable antirotavirus intestinal antibody of the IgG, but not IgA, isotype. After challenge, fourfold or greater increases in intestinal IgG antibody responses were observed in three rabbits, whereas all controls and all but one virus-vaccinated rabbit had an intestinal IgA antibody response. In contrast, vaccination of rabbits with one dose of SA11 followed by challenge at 21 DPV did not protect from challenge; no difference in the mean number of days of virus shedding between any of the vaccinated groups and controls was observed. A serologic, but not a mucosal, antibody response was observed after the one-dose vaccination regimen. Differences in serologic antibody titers were not observed between any of the one-dose virus-vaccinated groups. These data indicate that parenteral vaccination with two, but not one, doses of rotavirus in either Freund's adjuvant or aluminum phosphate can induce active protection from challenge.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inulin-derived adjuvants efficiently promote both Th1 and Th2 immune responses

There has been a recent resurgence of interest into new and improved vaccine adjuvants. This interest has been stimulated by the need for new vaccines to combat problematic pathogens such as SARS and HIV, and to counter potential bioterrorist attacks. A major bottleneck in vaccine development is the low immunogenicity of purified subunit or recombinant proteins, creating the need for safe human adjuvants with high potency. A major problem in the search for the ideal adjuvant is that adjuvants that promote cell-mediated (Th1) immunity (e.g. Freund's complete adjuvant) generally have unacceptable local or systemic toxicity that precludes their use in human vaccines. There is a need for a safe, non-toxic adjuvant that is able to stimulate both cell-mediated and humoral immunity. Inulin-derived adjuvants that principally stimulate the innate immune system through their ability to activate the alternative complement pathway have proven ability to induce both cellular and humoral immunity. With their excellent tolerability, long shelf-life, low cost and easy manufacture, they offer great potential for use in a broad range of prophylactic and therapeutic vaccines. Based on successful animal studies in a broad range of species, human trials are about to get underway to validate the use of inulin-based adjuvants in prophylactic vaccines against hepatitis B, malaria and other pathogens. If such trials are successful, then it is possible that inulin-derived adjuvants will one day replace alum as the adjuvant of choice in most human prophylactic vaccines.     

Comparison of immune and adverse effects induced by AdjuVac and Freund's complete adjuvant in New Zealand white rabbits (Oryctolagus cuniculus)

Though Freund's complete adjuvant effectively increases immune response to vaccines in various species, its potentially severe inflammatory effects have led many animal researchers to seek alternative immunological adjuvants. In a study of New Zealand white rabbits, the authors compared the immune and adverse effects of Freund's complete adjuvant with the effects of two formulations of AdjuVac, an immunological adjuvant previously developed by their group. All three adjuvants improved humoral immune response but also caused inflammation. Inflammatory reactions caused by AdjuVac, however, tended to be less severe than those caused by Freund's complete adjuvant.     

Cell recruitment and cytokines in skin mice sensitized with the vaccine adjuvants: saponin, incomplete Freund's adjuvant, and monophosphoryl lipid A

Vaccine adjuvants are substances associated with antigens that are fundamental to the formation of an intense, durable, and fast immune response. In this context, the use of vaccine adjuvants to generate an effective cellular immune response is crucial for the design and development of vaccines against visceral leishmaniasis. The objective of this study was to evaluate innate inflammatory response induced by the vaccine adjuvants saponin (SAP), incomplete Freund's adjuvant (IFA), and monophosphoryl lipid A (MPL). After a single dose of adjuvant was injected into the skin of mice, we analyzed inflammatory reaction, selective cell migration, and cytokine production at the injection site, and inflammatory cell influx in the peripheral blood. We found that all vaccine adjuvants were able to promote cell recruitment to the site without tissue damage. In addition, they induced selective migration of neutrophils, macrophages, and lymphocytes. The influx of neutrophils was notable at 12 h in all groups, but at other time points it was most evident after inoculation with SAP. With regard to cytokines, the SAP led to production of interleukin (IL)-2, IL-6, and IL-4. IFA promoted production of tumor necrosis factor (TNF)-α, interferon (IFN)-γ, IL-6, IL-17, IL-4, and IL-10. We also observed that MPL induced high production of IL-2, TNF-α, and IFN-γ, in addition to IL-6, IL-17, and IL-10. In peripheral blood, values of certain cell populations in the local response changed after stimulation. Our data demonstrate that the three vaccine adjuvants stimulate the early events of innate immune response at the injection site, suggesting their ability to increase the immunogenicity of co-administered antigens. Moreover, this work provides relevant information about elements of innate and acquired immune response induced by vaccine adjuvants administered alone.     

Protection against haemorrhagic septicaemia induced by vaccination of buffalo calves with an improved oil adjuvant vaccine

An experimental oil adjuvant vaccine was developed against haemorrhagic septicaemia, a disease of cattle and buffalo caused by Pasteurella multocida serotype B and E. Mineral oil, Mercol 52, was used as adjuvant together with Span 85 and Tween 85 as emulsifiers. The vaccine was evaluated by single dose intramuscular immunisation of 1–2 year old buffalo calves. IgG and IgM class antibodies were determined by ELISA. The group of animals immunised with the experimental oil adjuvant vaccine showed a high titre of the IgG class of antibodies measured at 300 days post vaccination. To compare the protective efficacy of the vaccine with the commonly used broth bacterin, another group of buffalo calves was immunised by broth bacterin. This group showed a low level of IgG antibodies. Protection was assessed by challenge with 10⁹ viable bacteria of P. multocida type B:2,5 administered subcutaneously, 250 days post vaccination. Animals vaccinated with the experimental oil adjuvant vaccine were fully protected. The other groups of animals, vaccinated with broth bacterin or used as control (non-vaccinated), developed symptoms of haemorrhagic septicaemia. A strong relationship between IgG but not IgM class antibody level and resistance to challenge was observed. The experiment demonstrated that the experimental oil adjuvant vaccine was superior to broth bacterin in providing protection against experimental haemorrhagic septicaemia in young buffalo calves beyond 250 days.     

Immunogenicity of a recombinant protein containing the Plasmodium vivax vaccine candidate MSP1(19) and two human CD4+ T-cell epitopes administered to non-human primates (Callithrix jacchus jacchus)

One of the most promising vaccine candidates against the erythrocytic forms of malaria is the 19 kDa C-terminal region of the merozoite surface protein 1 (MSP1(19)). As part of our studies aimed at the development of a Plasmodium vivax malaria vaccine, we characterized the immunogenic properties of a new bacterial recombinant protein containing the P. vivax MSP1(19) and two helper T-cell epitopes, the synthetic universal pan allelic DR epitope (PADRE) and a new internal MSP1 P. vivax epitope (DYDVVYLKPLAGMYK). We found that the recognition of His6MSP1(19)-DYDVVYLKPLAGMYK-PADRE was as good as the recognition of His6MSP1(19) indicating that the presence of the T-cell epitopes PADRE and DYDVVYLKPLAGMYK did not modify the MSP1(19) epitopes recognized by human IgG. The recombinant protein His6MSP1(19)-DYDVVYLKPLAGMYK-PADRE proved to be highly immunogenic in marmosets (Callithrix jacchus jacchus) when administered in incomplete Freund's adjuvant. However, when administered in other adjuvant formulations such as Quil A, CpG ODN 2006 or MPL/TDM, antibody titers to MSP1(19) were significantly lower. Among these three adjuvants, Quil A proved to be the most efficient one generating antibody titers significantly higher than the others. These results indicated that under the circumstances evaluated, adjuvants were key for the immunogenicity of the recombinant protein His6MSP1(19)-DYDVVYLKPLAGMYK-PADRE.     

Genomic maps of some strains within the Mycoplasma mycoides cluster

Genomic restriction maps for the small colony (SC) strains (PG1, KH3J, Gladysdale, and V5) of Mycoplasma mycoides subsp. mycoides (the agent of contagious bovine pleuropneumonia) and for Mycoplasma strain PG50 (classified as bovine serogroup 7), with respective sizes of 1,280, 1,280, 1,260, 1,230, and 1,040 kbp, were compared with the map (1,200 kbp) for a large colony strain (Y goat) of M. mycoides subsp. mycoides. The number and order of all mapped restriction sites were fully conserved in the SC genomes, as were the approximate positions of mapped loci. A number of these restriction sites in the Y genome and some, but fewer, in the PG50 genome appeared to be conserved. The SC and large colony strains shared conservation in the relative positions of the mapped loci, except for rpoC.     

Archaeosomes as adjuvants for combination vaccines

The present study evaluated the potential of archaesomes, prepared from the total polar lipids extracted from Methanobrevibacter smithii, as adjuvants for combination (multivalent) vaccines. Groups of Balb/c mice were immunized subcutaneously at day 0 and 21 with one of the following vaccines: trivalent vaccine formulated by the simultaneous co-encapsulation of bovine serum albumine (BSA), ovalbumin (OVA) and hen egg lysozyme (HEL) into archaeosomes (CEC vaccine); an univalent archaeosome vaccine (UVE vaccine) containing either BSA, OVA or HEL; or an admixture vaccine (AMC vaccine) consisting of the three UVE vaccines. Serum specific antibody (IgG + M) responses were determined at day 32, 112 and 203, and specific IgG1 and IgG2a responses were determined at day 112. Mice immunized with the CEC of AMC vaccine developed strong and sustained specific antibody responses to all three antigens at a magnitude similar to those seen in control mice immunized with UVE vaccines. Moreover, the serum BSA-, OVA-, and HEL-specific IgG1 and IgG2a levels in the CEC and AMC immunized mice were overall comparable to those of the UVE immunized control mice. Boosting CEC and AMC vaccinated mice with antigens alone at day 203 elicited strong antibody memory responses, comparable to those in the UVE vaccinated groups. These results show that archaeosomes could be used as adjuvants in developing combination vaccines.     

Comparison of protective effect of protein and DNA vaccines hsp90 in murine model of systemic candidiasis

Preventive vaccination by a hsp90-expressing DNA vaccine and recombinant hsp90 protein vaccine, both derived from the Candida albicans hsp90 using BALB-c mouse model of systemic candidiasis, was performed. Hsp90 mRNA was cloned from a clinical isolate of C. albicans, converted to cDNA and cloned into vaccination plasmid pVAX1. Two methods of DNA application were tested: intramuscular (i.m.) and intradermal (i.d.) injection. Recombinant protein was applied by i.d. injection with Freund's adjuvant; the control groups received PBS or Freund's adjuvant only. Mice were vaccinated and after 19 d re-vaccinated. After 3 weeks, the mice were challenged with the live C. albicans in a dose of 5 x 10(6) CFU per mouse. After the challenge, the mice vaccinated i.d. with DNA vaccine survived for 39 and 64% longer compared to those receiving Freund's adjuvant and/or PBS, respectively. The i.m. application of the DNA vaccine did not provide any significant protectivity. The serum level of anti-candida-hsp90 serum IgG antibodies correlated with the survival rate in both i.d. protein and DNA vaccination approaches. We stressed the importance of specific humoral immunity in the mouse model of systemic candidiasis.     

The immune response of the brown trout, Salmo trutta, L. to MS2 bacteriophage: immunogen concentration and adjuvants

The humoral antibody response to single intraperitoneal inoculations of MS2 bacteriophage was followed in Salmo trutta using a 50% bacteriophage neutralisation litre ( sd ₅₀) method. Immune memory was observed with enhancement of both the time and level of antibody production, though an initial suppression was observed 7 days after secondary inoculation. Antibody titres were related to MS2 concentration, and adjuvants were found to enhance the response. An increased enhancement of antibody production by Freund's complete adjuvant over that of incomplete adjuvant was observed.     

Characterization of cross protection of Swine-Origin Influenza Virus (S-OIV) H1N1 and reassortant H5N1 influenza vaccine in BALB/c mice given a single-dose vaccination

Background

Influenza virus has antigen drift and antigen shift effect, vaccination with some influenza vaccine might not induce sufficient immunity for host to the threat of other influenza virus strains. S-OIV H1N1 and H5N1 influenza vaccines in single-dose immunization were evaluated in mice for cross protection to the challenge of A/California/7/2009 H1N1 or NIBRG-14 H5N1 virus.

Results

Both H1N1 and H5N1 induced significant homologous IgG, HAI, and microneutralization antibody responses in the mice, while only vaccines plus adjuvant produced significant heterogeneous IgG and HAI antibody responses. Both alum and MPLA adjuvants significantly reduced the S-OIV H1N1 vaccine dose required to elicit protective HAI antibody titers from 0.05 μg to 0.001 μg. Vaccines alone did not protect mice from challenge with heterogeneous influenza virus, while H5N1 vaccine plus alum and MPLA adjuvants did. Mouse body weight loss was also less significant in the presence of adjuvant than in the vaccine without adjuvant. Furthermore, both H1N1 and H5N1 lung viral titers of immunized mice were significantly reduced post challenge with homologous viruses.

Conclusion

Only in the presence of MPLA adjuvant could the H5N1 vaccine significantly reduce mouse lung viral titers post H1N1 virus challenge, and not vice versa. MPLA adjuvant induced cross protection with a single dose vaccination to the challenge of heterogeneous influenza virus in mice. Lung viral titer seemed to be a better indicator compared to IgG, neutralization antibody, and HAI titer to predict survival of mice infected with influenza virus.     

Immunisation of channel catfish,Ictalurus punctatus,with Ichthyophthirius multifiliis immobilisation antigens elicits serotype-specific protection

Surface immobilisation antigens (i-antigens) were purified from two strains of Ichthyophthirius multifiliis (NY1 and G5) that represent different i-antigen serotypes, namely A and D, respectively. The efficacy of the purified antigens as subunit vaccines was then tested in challenge studies using parasites of the homologous or heterologous serotype. Three groups of juvenile channel catfish (70 animals per group) were immunised with i-antigens from either the G5 or NY1 isolates, or with bovine serum albumin (BSA) as a control. Proteins were injected intraperitoneally (i.p.) at a dose of 10 microg/fish with complete Freund's adjuvant on day 1, followed by a second injection in incomplete Freund's adjuvant on day 15. Fish immunised with the purified i-antigens developed high titres of serum immobilising antibodies whereas sera from BSA-injected control fish did not. Fish antisera immobilised parasites of the homologous, but not the heterologous strain, and recognised the corresponding i-antigens on Western blots run under non-reducing conditions. On day 36, each group was divided into two subgroups (n=30). One subgroup was challenged with G5 parasites, and the other was challenged with NY1 parasites. When challenged with G5 parasites, 70% of fish immunised with the G5 i-antigens survived. When challenged with NY1 parasites, 33.3% of fish immunised with the NY1 i-antigens survived. All BSA-injected control fish died, as did all fish injected with the purified antigens and challenged with the non-homologous parasite strain. Statistical analyses indicated significant differences among test and control groups with regard to the mean days to death (MDD). While the results of these studies clearly support a role for i-antigens in protection, active immunity in response to natural infection is not serotype-specific. The utility of i-antigens, as well as the existence of other potential vaccine candidates for the prevention of 'white-spot' disease, are discussed.