Inducement of Respiratory Mucosal Immunogenicity for Nasal Vaccine Due to Amine- and Amide-Inactivation of Parainfluenza Virus Type 1, Sendai

The protective effects in mice by nasal vaccination of amine- and amide-inactivated Sendai viruses were investigated by a contact exposure experiment, immunofluorescent examination of the entire respiratory tract, and checking the serum HI antibody development. Of 10 monoamines, ethanolamine and 2-methoxyethylamine vaccines induced complete protection, and methylamine, ethylamine, n-propylamine, n-butylamine, 2-ethoxyethylamine, diethylamine and triethylamine vaccines brought about almost complete protection or lower respiratory infection. The methoxyamine-treated mouse conferred the least protection. Of 5 diamines, 1, 3-diaminopropane vaccine inhibited completely the infection, but hydrazine, ethylenediamine, putrescine, and cadaverine vaccines produced regional infection. Two polyamines, spermine and spermidine, did not inactivate the virus. Of 4 amides, only semicarbazide vaccine conferred complete mucosal defense, while acetamide, propionamide, and isonicotinic acid hydrazide vaccines lead to regional infection. Serum HI titers developed by vaccination were low on the whole, following their slight rise, fall or maintenance postexposure. In effect, the 4 vaccines inactivated by a best-suited interstrand crosslink between phosphate groups in helix of viral RNA brought about the strongest protection, and showed the necessity of a definite length of molecules for inactivants.     

Functional Differences of Dyes in Inducing Respiratory Immunogenicity by Azo-, Thiazole-, Quinoline-, Reactive-, and Naphthol-Dye-Inactivated Sendai Viruses

The prophylactic effects by mouse nasal inoculation of 31 kinds of organic dye-inactivated Sendai viruses were investigated by contact exposure experiment that used mouse nasally infected with 10^5,8 EID₅₀, immunofluorescent examination of the entire respiratory tract, and check of rise of serum HI titer postexposure. The relative merits of the dye-structures for inciting nasal immunogenicity were determined. Of 14 azo-dye-inactivated vaccines, only azo blue- and amido black 10B treated ones brought about nearly complete protection, while the other 5 dye-groups provided partial protection and the remaining 7 dye-groups the least protection. Of 6 thiazole dye-vaccines, primuline-, thioflavine S-, and thioflavine T-vaccines induced complete or almost complete protection, and the others moderate or the least protection. With 3 quinolinevaccines, both pinacyanol- and quinaldine red-inactivated ones provided complete protection, but not with quinoline blue-vaccine. Of 4 reactive dye-vaccines, both cibacron brilliant yellow 3G-P-and reactive blue 4-treated ones brought about nearly complete protection, but the remaining 2 vaccines induced regional infection. Nevertheless, all 4 naphthol group (AS, AS-BS, AS-BI, and AS-MX)-treated vaccines yielded complete or almost complete protection. The thirteen most effective vaccine groups suppressed marked rise of high or low serum HI titers developed through nasal vaccination postexposure. In short, specified dyestuffs having a great affinity for cellulosic fibers, evidently incited mucosal immunogenicity, probably by major union of the dyes to viral core ribose.     

Protective effects of nasal immunization in mice with various kinds of inactivated Sendai virus vaccines

The immunoprophylactic effects of nasal vaccination with 13 different kinds of inactivated Sendai virus vaccines were compared by contact exposure to infector mice. Efficacies of the vaccines were evaluated on the basis of the presence of virus-infected cells by immunofluorescent examination of the entire respiratory tract, including the nasal mucosa. A single or double inoculations of B-propiolactone (0.5%)-vaccine promoted the infection in the respiratory tract, particularly in the nasal mucosa, whereas three inoculations of B-propiolactone (0.2%)-vaccine provided considerable protection throughout the respiratory tract with only slight development of serum HI titer. Formalin (0.1%)-vaccine and UV irradiated-vaccine strongly protected the nasal mucosa from infection, but did not sufficiently safeguard the lower respiratory tract even with three vaccinations despite adequate development of serum antibody. Nearly complete protection of the entire respiratory tract was induced with six to eight inoculations of a vaccine treated excessively with both UV rays and 1% formalin, without significant development of serum antibody. Out of eight thermal vaccines, five (inactivated at 23 C, 30 C, 37 C and 7 C, and 30 C and 7 C) provided strong protection against infection when inoculated three times. The others inactivated at higher temperatures (37 C, 50 C, or 60 C) were not so protective. High serum HI titers developed, on the whole, with the drop in the temperature required for inactivating the virus. In eight immune mouse groups in which infection was strongly suppressed in the entire respiratory tract, most of the mice harbored less than 50 viral antigen-positive cells in their nasal mucosa in the postexposure period. The number of the cells was assumed to be a useful criterion for evaluation of vaccine efficacy.     

Selection of the most efficacious of twenty-two inactivated Sendai virus nasal vaccines by determination of the protection index in mice.

BACKGROUND AND PURPOSE: Sendai virus nasal vaccines inactivated with various chemicals induce complete protection against contact-challenge exposure with the Nagoya strain. The study reported here was to reevaluate the efficacy of the inactivants by determining the protective index (PI) in mice, using the more virulent MN strain. METHODS: Mice were given each of 22 inactivated vaccines intranasally three times. After challenge exposure with 10(-2) to 10(6) MID50 of virus, infection of cells of the respiratory tract was determined by immunofluorescence. RESULTS: Twelve vaccines induced PI > or = 2.0 in the nasal mucosa and were classified as group 1. The first half of the preceding vaccines that induced PI > or = 3.2 in the larynx were classified subgroup a, and the rest were classified subgroup b. Of the other 10 vaccines, 6 that induced PI < or = 2.0 in the larynx and 4 that induced intermediate PI in the nasal mucosa and larynx were ranked as groups 3 and 2, respectively; PI of the trachea decreased by numeric order of groups. Serum hemagglutination inhibition titer induced by intranasal vaccination was low in general. CONCLUSION: On the basis of PI values, 6 of the 22 nasal vaccines provided the strongest defense in the respiratory tract.     

Comparative evaluation on mouse nasal immunogenicity of arylmethane-, xanthene-, quinone-imine-, and acridine-dye-inactivated Sendai virus vaccines

Twenty-seven kinds of organic dye-inactivated Sendai virus vaccines were prepared by treatment in dark at 23 C for 2 months or more, and selected with the high HA titers as a guide. Their nasal immunogenicities were examined in mice by contact infection and immunofluorescent method, and the relative merits of the dye-inactivants were determined. The strongest protection was elicited with acriflavine-, auramine O-, eosin Y-, neutral red-, night blue-, patent blue V-, thymol blue-, uranin-, and xylene cyanol FF-treated vaccines. Middling protective efficacy was induced by use of erio green B-, malachite green-, methyl green-, proflavine-, pyronin B-, and thionin-inactivated vaccines. Dye-inactivated vaccines that resulted in the weakest protection were Bindschedler's green-, bromothymol blue-, erythrosin B-, ethyl violet-, gallein-, light green SF yellowish-, methyl violet-, new methylene blue N-, phenol red-, rhodamine 6G-, spirit blue- and victoria blue B-treated ones. Serum HI titers developed by nasal vaccination were variable, and rose still more in most vaccinated groups postexposure. Elicitation of the most effective nasal immunogenicity in dye-inactivated vaccines appeared to depend on selective modification of capsid protein or ribose in viral core with dyes possessing definite functions, despite the different molecular structures.     

Oral administration of cholera toxin-Sendai virus conjugate potentiates gut and respiratory immunity against Sendai virus

Successful oral immunization to prevent infectious diseases in the gastrointestinal tract as well as distant mucosal tissues may depend on the effectiveness of an Ag to induce gut immune responses. We and others have previously reported that cholera toxin possesses strong adjuvant effects on the gut immune response to co-administered Ag. To explore further adjuvant effects of cholera toxin, the holotoxin or its B subunit was chemically cross-linked to Sendai virus. The resulting conjugates, which were not infectious, were evaluated for their capacity to induce gut immune responses against Sendai virus after oral administration to mice. Conjugating cholera toxin to virus significantly enhanced the adjuvant activity of cholera toxin compared to simple mixing. Cholera toxin B subunit, however, did not show an adjuvant effect either by itself or conjugated with the virus. Oral administration of the Sendai virus-cholera toxin conjugate was also able to prime for protective anti-viral responses in the respiratory tract. Mice that were orally immunized with the conjugate and intra-nasally boosted with inactivated virus alone showed virus-specific IgA titers in nasal secretions that correlated with protection against direct nasal challenge with live Sendai virus. For comparison, s.c. immunization was also studied. Systemic immunization with the virus-cholera toxin conjugate induced virus-specific antibody responses in serum as well as in the respiratory tract but failed to protect the upper respiratory tract against virus challenge. Systemic immunization plus an intra-nasal boost did, however, confer a variable degree of protection to the upper respiratory tract, which correlated primarily with bronchoalveolar lavage (lung) antibody titers.     

The adjuvant effect of Sophy β-glucan to the antibody response in poultry immunized by the avian influenza A H5N1 and H5N2 vaccines

Avian influenza virus vaccines produced in oil-emulsified inactivated form with antigen content of at least 160 hemagglutinin units (HAU) induced immunity in birds. However, in addition to enhancing the effect of the adjuvant(s), other additional supplemented biological compounds included in inactivated vaccines could produce higher levels of antibody. We examined in chickens, Vietnamese ducks, and muscovy ducks the adjuvant effect of Sophy β-glucan (SBG), a β-1,3-1,6 glucan produced by the black yeast Aureobasidium pollulans strain AF0-202, when administered with an avian influenza H5 subtype vaccine. In Experiment 1, 40 chickens (ISA Brown hybrid), allocated to four groups of ten each, were immunized with Oil-H5N1(VN), Oil-H5N1(CN), Oil-H5N2(CN), and saline (control group), respectively. In Experiment 2, chickens (ISA Brown hybrid), muscovy ducks (French hybrid), and Vietnamese ducks (indigenous Vietnamese) were used to further assess the effect of SBG on immunogenicity of the Oil-H5N1(VN) Vietnamese vaccine. ELISA and hemagglutination inhibition (HI) assays were used to assess the antibody response. The H5 subtype vaccines initiated significantly higher immune responses in the animals dosed with SBG, with 1.0-1.5 log2 higher HI titers and 10-20% ELISA seroconversion, compared with those not dosed with β-glucan. Notably, some of the animals dosed with SBG induced HI titers higher than 9.0 log2 following boosting immunization. Taken together, our serial studies indicated that SBG is a potential effector, such as enhancing the immune response to the H5 vaccines tested.     

Live Attenuated Influenza Vaccine Provides Superior Protection from Heterologous Infection in Pigs with Maternal Antibodies without Inducing Vaccine-Associated Enhanced Respiratory Disease

Control of swine influenza A virus (IAV) in the United States is hindered because inactivated vaccines do not provide robust cross-protection against the multiple antigenic variants cocirculating in the field. Vaccine efficacy can be limited further for vaccines administered to young pigs that possess maternally derived immunity. We previously demonstrated that a recombinant A/sw/Texas/4199-2/1998 (TX98) (H3N2) virus expressing a truncated NS1 protein is attenuated in swine and has potential for use as an intranasal live attenuated influenza virus (LAIV) vaccine. In the present study, we compared 1 dose of intranasal LAIV with 2 intramuscular doses of TX98 whole inactivated virus (WIV) with adjuvant in weanling pigs with and without TX98-specific maternally derived antibodies (MDA). Pigs were subsequently challenged with wild-type homologous TX98 H3N2 virus or with an antigenic variant, A/sw/Colorado/23619/1999 (CO99) (H3N2). In the absence of MDA, both vaccines protected against homologous TX98 and heterologous CO99 shedding, although the LAIV elicited lower hemagglutination inhibition (HI) antibody titers in serum. The efficacy of both vaccines was reduced by the presence of MDA; however, WIV vaccination of MDA-positive pigs led to dramatically enhanced pneumonia following heterologous challenge, a phenomenon known as vaccine-associated enhanced respiratory disease (VAERD). A single dose of LAIV administered to MDA-positive pigs still provided partial protection from CO99 and may be a safer vaccine for young pigs under field conditions, where dams are routinely vaccinated and diverse IAV strains are in circulation. These results have implications not only for pigs but also for other influenza virus host species.     

Pathogenicity and vaccine efficacy of different clades of Asian H5N1 avian influenza A viruses in domestic ducks

Waterfowl represent the natural reservoir of all subtypes of influenza A viruses, including H5N1. Ducks are especially considered major contributors to the spread of H5N1 influenza A viruses because they exhibit diversity in morbidity and mortality. Therefore, as a preventive strategy against endemic as well as pandemic influenza, it is important to reduce the spread of H5N1 influenza A viruses in duck populations. Here, we describe the pathogenicity of dominant clades (clades 1 and 2) of H5N1 influenza A viruses circulating in birds in Asia. Four representatives of dominant clades of the viruses cause symptomatic infection but lead to different profiles of lethality in domestic ducks. We also demonstrate the efficacy, cross-protectiveness, and immunogenicity of three different inactivated oil emulsion whole-virus H5 influenza vaccines (derived by implementing reverse genetics) to the viruses in domestic ducks. A single dose of the vaccines containing 1 μg of hemagglutinin protein provides complete protection against a lethal A/Duck/Laos/25/06 (H5N1) influenza virus challenge, with no evidence of morbidity, mortality, or shedding of the challenge virus. Moreover, two of the three vaccines achieved complete cross-clade or cross-subclade protection against the heterologous avian influenza virus challenge. Interestingly, the vaccines induce low or undetectable titers of hemagglutination inhibition (HI), cross-HI, and/or virus neutralization antibodies. The mechanism of complete protection in the absence of detectable antibody responses remains an open question.     

Evaluation of the Efficacy and Cross-Protectivity of Recent Human and Swine Vaccines against the Pandemic (H1N1) 2009 Virus Infection

The current pandemic (H1N1) 2009 virus remains transmissible among humans worldwide with cases of reverse zoonosis, providing opportunities to produce more pathogenic variants which could pose greater human health concerns. To investigate whether recent seasonal human or swine H1N1 vaccines could induce cross-reactive immune responses against infection with the pandemic (H1N1) 2009 virus, mice, ferrets or mini-pigs were administered with various regimens (once or twice) and antigen content (1.77, 3.5 or 7.5 µg HA) of a-Brsibane/59/07, a-CAN01/04 or RgCA/04/09xPR8 vaccine. Receipt of a-CAN01/04 (2-doses) but not a-Brisbane/59/07 induced detectable but modest (20–40 units) cross-reactive serum antibody against CA/04/09 by hemagglutinin inhibition (HI) assays in mice. Only double administration (7.5 µg HA) of both vaccine in ferrets could elicit cross-reactivity (30–60 HI titers). Similar antigen content of a-CAN01/04 in mini-pigs also caused a modest ∼30 HI titers (twice vaccinated). However, vaccine-induced antibody titers could not suppress active virus replication in the lungs (mice) or virus shedding (ferrets and pigs) of immunized hosts intranasally challenged with CA/04/09. Furthermore, neither ferrets nor swine could abrogate aerosol transmission of the virus into naïve contact animals. Altogether, these results suggest that neither recent human nor animal H1N1 vaccine could provide complete protectivity in all animal models. Thus, this study warrants the need for strain-specific vaccines that could yield the optimal protection desired for humans and/or animals.     

Serologic studies of acute respiratory infections in military personnel.

The advantages, disadvantages, and uses of serological epidemiology are discussed in relation to acute respiratory infections in military personnel. The prevalence of antibody reflects both current and past experience with respiratory agents and is a measure of susceptinility. Incidence data calculated by testing two serial serum samples, on entry and discharge from the service, has indicated high influenza and mycoplasma pneumoniae rates in South American recruits and low rates of adenovirus and parainfluenza infections. Serologic analysis of reinfection rates showed high protection against influenza infections at HI antibody levels of over 1:40, against adenovirus infections at neutralizing titers of 1:5, and against M. pneumoniae infections at TRI antibody levels over 1:8. Antibody responses persisting at least 7 mo following immunization were demonstrated in 70% of 428 vaccinated young adults for A2 antigen and 20% for influenza B antigen. No relation of ABO blood groups to respiratory infection was found. The lack of myxovirus infections in four Polaris submarines is presented.     

Comparison of dimethyl dioctadecyl ammonium bromide, Freund's complete adjuvant and mineral oil for induction of humoral antibodies, cellular immunity and resistance to Newcastle disease virus in chickens

Abstract Dimethyl diotadecyl ammonium bromide (DDA), a lipophilic quaternary amine, was evaluated in adult chickens for potentiation of immunological responses to subcutaneously administered inactivated Newcastle disease virus (NDV) vaccines. DDA enhanced humoral and cell-mediated immune (CMI) responses to levels which were significantly higher than those induced by the vaccine alone The haemagglutination inhibition antibody titers induced by DDA were slightly lower than those induced by mineral oil although neutralizing antibody titers seemed to be higher. DDA induced strong CMI (DTH and lymphocyte proliferation) responses, more than those induced by Freund's complete adjuvant and mineral oil. Both DDA and mineral oil induced comparable high levels of protection to challenge doses of 200 000 LD₅₀ per chicken. No toxic effects or local tissue damage were observed in any of the inoculated chickens.     

Serum and mucosal immune responses to an inactivated influenza virus vaccine induced by epidermal powder immunization

Both circulating and mucosal antibodies are considered important for protection against infection by influenza virus in humans and animals. However, current inactivated vaccines administered by intramuscular injection using a syringe and needle elicit primarily circulating antibodies. In this study, we report that epidermal powder immunization (EPI) via a unique powder delivery system elicits both serum and mucosal antibodies to an inactivated influenza virus vaccine. Serum antibody responses to influenza vaccine following EPI were enhanced by codelivery of cholera toxin (CT), a synthetic oligodeoxynucleotide containing immunostimulatory CpG motifs (CpG DNA), or the combination of these two adjuvants. In addition, secretory immunoglobulin A (sIgA) antibodies were detected in the saliva and mucosal lavages of the small intestine, trachea, and vaginal tract, although the titers were much lower than the IgG titers. The local origin of the sIgA antibodies was further shown by measuring antibodies released from cultured tracheal and small intestinal fragments and by detecting antigen-specific IgA-secreting cells in the lamina propria using ELISPOT assays. EPI with a single dose of influenza vaccine containing CT or CT and CpG DNA conferred complete protection against lethal challenges with an influenza virus isolated 30 years ago, whereas a prime and boost immunizations were required for protection in the absence of an adjuvant. The ability to elicit augmented circulating antibody and mucosal antibody responses makes EPI a promising alternative to needle injection for administering vaccines against influenza and other diseases.     

Construction and characterization of a live, attenuated apxIICA inactivation mutant of Actinobacillus pleuropneumoniae lacking a drug resistance marker

The apxIIC gene of Actinobacillus pleuropneumoniae serotype 7 was inactivated by homologous recombination using a sucrose counter-selectable marker system, resulting in a mutant strain that had no antibiotic resistance marker and expressed an inactivated ApxII toxin. The safety and immunogenicity of the mutant were evaluated in mice. The mutant strain caused no adverse effects in mice at doses up to 2 x 10(9) CFU via the intraperitoneal route while the parental strain induced total mortality at a dose of 2 x 10(7) CFU. Mice vaccinated intraperitoneally with the mutant strain had 100% and 70% protection against homologous (serotype 7) or heterologous (serotype 1, 3) challenge with A. pleuropneumoniae, respectively. The A. pleuropneumoniae mutant strain HB04C- and the counterselection method used in the study show promise in developing effective live vaccines for porcine pleuropneumonia and for other infections diseases of the respiratory system.     

Antibody production in rats vaccinated with inactivated Sendai virus (author's transl)

Adult male and female rats were inoculated with tween 80-ethylether-formalin-ultraviolet inactivated Sendai virus (Sv-V) and examined for the production of hemagglutination inhibition (HI) antibody. There were no significant differences in the antibody titers between males and females, and among the various routes of inoculation except for the intranasal which was not effective. The antibody became detectable 7 days after a single inoculation with 10(5) HAU of Sv-V. The antibody titer, which had its peak 21 days after the inoculation, persisted for 200 days and declined gradually thereafter. The HI antibody titers were correlated with inoculated Sv-V doses and a predominant booster reaction with the vaccine was observed. Maternal antibodies were detected in sucklings born to dams hyperimmunized with the vaccine. The titers were similar to those of the dams until 3 weeks after birth but declined rapidly after weaning at 4-week-old. The titers of fetuses and neonates before suckling were significantly lower than those of the sucklings.     

Combined oral/nasal immunization protects mice from Sendai virus infection

Based on the concept of a common mucosal immune system wherein mucosal associated lymphocytes traffic among the various mucous membranes, the murine gastrointestinal tract was immunized with Sendai virus antigens in order to elicit a virus-specific immune response in the respiratory tract. Multiple intragastric (oral) administration of live or killed Sendai virus induced IgA and IgG antiviral antibodies in both gastrointestinal secretions and serum. When cholera toxin as an adjuvant was included along with virus, gut IgA and IgG as well as serum IgA responses were enhanced. Antiviral antibodies induced in respiratory secretions by oral killed virus plus cholera toxin, however, were variable and protection from virus challenge was not demonstrated. Significantly higher levels of respiratory antiviral antibodies were induced if immunization with oral killed Sendai virus/cholera toxin was combined with intranasal administration of small amounts of killed virus. The combined immunization also resulted in protection of both the upper and lower respiratory tracts from virus infection. Protection of the upper respiratory tract was correlated with the presence of IgA antiviral antibodies in nasal washings. On the other hand, protection of the lower respiratory tract was correlated with IgG antiviral antibodies in bronchoalveolar lavage fluids. Immunization with intranasal killed virus alone conferred partial protection to the lower respiratory tract and no protection to the upper respiratory tract. Thus, oral immunization with killed virus antigen could prime for a protective immune response in the murine respiratory tract and this protective response included IgA antibodies.     

Antibody responses to intradermal or intramuscular MF59-adjuvanted influenza vaccines as evaluated in elderly institutionalized volunteers during a season of partial mismatching between vaccine and circulating A(H3N2) strains

Background

The age-related weakening of the immune system makes elderly subjects less responsive to influenza vaccination. In the last years, two “enhanced vaccines” were licensed for individuals aged ≥65 years, one being a subunit vaccine (Fluad®) containing the MF59 adjuvant administered intramuscularly (IM-MF59) and the other one a split non-adjuvanted vaccine administered intradermally (Intanza® 15mcg) (ID). In the present study, we evaluated and compared the antibody responses against the three vaccine antigens and heterovariant A(H3N2) circulating viruses induced by IM-MF59 and ID influenza vaccines in 80 elderly institutionalized volunteers (40 per group) during the Winter season 2011–2012.

Results

Hemagglutination inhibiting (HI) antibody titers were assessed in blood samples collected before, 1 and 6 months after vaccination. One month after vaccination both the IM-MF59 and ID vaccines induced increases in HI titers against all the three vaccine strains. The results in the two groups were similar against the A(H3N2) and A(H1N1) strains. Responses against the B strain typically tended to be higher after ID than IM-MF59, yet both vaccines stimulated lower responses against the B strain than against the two A strains. The two vaccines induced favorable results also against four epidemic drifted A(H3N2) viruses circulating in Winter 2011–2012. Six months after vaccination, the HI titers decreased in both groups.

Conclusion

The responses induced by IM-MF59 and ID vaccines in institutionalized elderly people were similar against the A(H3N2) and A(H1N1) strains but frequently higher, for the ID, against the B strain. The two vaccines induced positive responses against drifted A(H3N2) circulating viruses.

     

禽呼肠孤病毒感染对鸡法氏囊及免疫应答的影响

研究LY株禽呼肠孤病毒(ARV)感染1日龄SPF鸡后对法氏囊发育影响,对传染性法氏囊病毒(IBDV)、禽流感病毒(AIV)、新城疫病毒(NDV)疫苗免疫诱发的抗体的影响,及对强毒株IBDV致病作用的影响。结果表明,LY株ARV感染1日龄SPF鸡可引起法氏囊萎缩和部分淋巴细胞减少,但对增重及AIV和NDV疫苗免疫后抗体滴度却没有显著影响。ARV感染可降低弱毒IBDV疫苗免疫后的抗体反应,但对随后IBDV强毒株攻毒的抵抗力却与对照鸡无显著差异。经IBDV弱毒疫苗免疫后,再接种强毒株IBDV,不会引起死亡,但却仍能显著抑制对AIV、NDV疫苗免疫后的抗体滴度。然而,对于1~7日龄经ARV感染的鸡,IBDV强毒的这种免疫抑制作用又显著低于未经ARV感染的对照鸡。     

Induction of heterosubtypic immunity to influenza virus by intranasal immunization

Recovery from live influenza virus infection is known to induce heterosubtypic immunity. In contrast, immunity induced by inactivated vaccines is predominantly subtype specific. In this study, we investigated the heterosubtypic protective immunity induced by inactivated influenza virus. Intranasal immunization of mice with inactivated influenza virus A/PR8 (H1N1) provided complete protection against the homologous virus and a drift virus within the same subtype, A/WSN (H1N1), but not against the heterosubtypic virus A/Philippines (H3N2). However, coadministration of inactivated virus with cholera toxin as an adjuvant conferred complete heterosubtypic protection, without observed illness, even under conditions of CD4⁺ or CD8⁺ T-cell depletion. Analysis of immune correlates prior to challenge and postchallenge indicated that humoral immune responses with cross-neutralizing activity in lungs and in sera play a major role in conferring protective immunity against heterosubtypic challenge. This study has significant implications for developing broadly cross-reactive vaccines against newly emerging pathogenic influenza viruses.     

Temperature-dependent protection against Ichthyophthirius multifiliis following immunisation of rainbow trout using live theronts

Rainbow trout Oncorhynchus mykiss Walbaum, 1792 fingerlings were vaccinated by intraperitoneal (i.p.) injection using live theronts of the skin parasitic ciliate Ichthyophthirius multifiliis Fouquet, 1876 at 2 temperatures (12 and 20 degrees C), and protection against challenge infections was subsequently evaluated by bath exposure to live theronts. Vaccination conferred a relative protection (evaluated as the decrease in the number of established theronts) at 12 degrees C and almost complete immunity at 20 degrees C. Significantly increased immobilisation titers (using plasma immobilisation of live theronts) were found in immunised fish at Week 2 and 4 post-vaccination. Lysozyme activity of plasma from vaccinated fish increased from Week 1 to 4. Both immobilisation titers and lysozyme activity were significantly higher at 20 degrees C. This study demonstrated that live theronts are good candidates for an antigen source for development of effective vaccines against white spot disease in this fish host, and further indicated that the protection of rainbow trout against I. multifiliis infection is highly temperature dependent and may be associated with both adaptive and innate response mechanisms.