Multistrain influenza protection induced by a nanoparticulate mucosal immunotherapeutic.

All commercial influenza vaccines elicit antibody responses that protect against seasonal infection, but this approach is limited by the need for annual vaccine reformulation that precludes efficient responses against epidemic and pandemic disease. In this study we describe a novel vaccination approach in which a nanoparticulate, liposome-based agent containing short, highly conserved influenza-derived peptides is delivered to the respiratory tract to elicit potent innate and selective T cell-based adaptive immune responses. Prepared without virus-specific peptides, mucosal immunostimulatory therapeutic (MIT) provided robust, but short-lived, protection against multiple, highly lethal strains of influenza in mice of diverse genetic backgrounds. MIT prepared with three highly conserved epitopes that elicited virus-specific memory T-cell responses but not neutralizing antibodies, termed MITpep, provided equivalent, but more durable, protection relative to MIT. Alveolar macrophages were more important than dendritic cells in determining the protective efficacy of MIT, which induced both canonical and non-canonical antiviral immune pathways. Through activation of airway mucosal innate and highly specific T-cell responses, MIT and MITpep represent novel approaches to antiviral protection that offer the possibility of universal protection against epidemic and pandemic influenza.

Humoral and cell-mediated immune responses of humans to inactivated influenza vaccine with or without QS21 adjuvant.

To evaluate humoral (antibody) and cell mediated immune (CMI) responses, 30 healthy young adults were either given inactivated influenza vaccine with or without QS21 adjuvant. Vaccination site pain and postvaccination myalgias were greater in the QS21 group. Serum antibody increases occurred in 73-93% of subjects for each vaccine and antigen at 2 weeks and 4 weeks but frequencies and mean titers for the two vaccines were not different. No differences in T cell cytotoxicity were detected for either vaccine for influenza A or B infected cells. IFN-gamma for both vaccine groups was increased in supernates after 3 days but not 7 days of stimulation in the cytotoxicity tests; amounts for the two vaccines were similar. To further evaluate CMI, remaining PBMCs were stimulated overnight with cells infected with each vaccine strain; an increase in spot forming cells (sfc) for Granzyme B and IFN-gamma was found for all subjects and in 51 of 54 sfc tests. A slightly higher response in the Gran B test for QS21 subjects was suggested, but no clear immune response advantage was identified among healthy adults for QS21 adjuvanted influenza vaccine.

Apoptosis and reduced influenza A virus specific CD8+ T cells in aging mice.

Some studies have reported increased apoptosis in CD8(+) T cells from aged mice. We previously demonstrated diminished virus-specific CD8(+) cytotoxic T lymphocyte (CTL) activity in aged mice in comparison to young mice. The present study investigated the role of apoptosis in age-related influenza virus-specific CD8(+) CTL deficiency. Splenocytes from influenza-primed aged and young mice were stimulated in vitro with virus. The CD8(+) T cell/total lymphocyte ratios correlated with CTL activity and were significantly decreased and increased in aged and young mice, respectively. Fas, FasL, TNF-alpha and TNFR-p55 expression, measured by flow cytometry, ELISA and/or RT-PCR, were significantly elevated in aged mice. Apoptotic CD8(+) T cells (Annexin V binding) were also elevated in aged mice. IL-12 treatment increased CD8(+) CTL activity and IFN-gamma production but did not affect apoptosis. Thus, apoptosis may contribute to reduced influenza virus-specific CD8(+) T cell frequency, CTL deficiency and increased influenza disease in aging.

Independent and synergistic effects of interleukin-18 and interleukin-12 in augmenting cytotoxic T lymphocyte responses and IFN-gamma production in aging.

Aged mice exhibit diminished CD8(+) cytotoxic T lymphocyte (CTL) response to influenza virus. Previously, interleukin-12 (IL-12) was shown to partially restore in vitro influenza virus-specific CD8(+) CTL activity and interferon-gamma (IFN-gamma) production in aged mice. The present study investigated IL-18 production and its ability to collaborate with IL-12 to enhance these responses to the levels of young mice. IL-18 protein production and mRNA expression in influenza virus-specific CTL from aged mice were higher than from young mice. In contrast, IL-18 receptor (IL-18R) mRNA expression was significantly reduced in CD8(+) CTL from aged mice. Generation of CTL in the presence of IL-12 alone caused a significant increase in IFN-gamma production in both old and young mice. IL-18 treatment alone significantly increased IFN-gamma in CTL from young but not old mice. However, a combination of IL-18 and IL-12 significantly increased IFN-gamma in both old and young mice. IL-18 and IL-12, either alone or in combination, stimulated significant influenza virus-specific cytotoxicity in both old and young mice, but no significant synergistic effect was observed. These results represent an initial demonstration of downregulated IL-18R expression in aging mice and are consistent with age-related cytotoxic T lymphocyte 1 (Tc1) deficiency. Potentially, IL-18 and IL-12 can augment IFN-gamma production and reverse CD8(+) CTL deficiency in aging, independently or synergistically.

HLA-restricted CD8+ cytotoxic T lymphocyte, interferon-gamma, and interleukin-4 responses to respiratory syncytial virus infection in infants and children.

CD8+ cytotoxic T lymphocyte (CTL) activity, interferon (IFN)-gamma, and interleukin (IL)-4 production were evaluated in a blinded manner among respiratory syncytial virus (RSV)-infected newborns and their mothers for 3 epidemic seasons. Most mothers (80%) exhibited RSV-specific CD8+ CTL activity. Twenty (80%) of the 26 infants exhibited significant RSV-specific CTL activity during or after their first RSV season. CTL frequency increased with RSV infection rate, reaching 75% by the end of the third season. Most infants who shed virus (75%) had a medically attended lower respiratory tract disease (LRD). In the first year, RSV-infected infants (virus culture and antibody increase) were more likely to develop CTL activity (10 of 13) than were uninfected infants (1 of 5; P=.02). Infants with CTL activity in the first year were less likely to have an LRD in the second year. CD8+ CTL levels correlated positively with IFN-gamma (P<.001) and inversely with IL-4 (P=.03). Contribution of CD8+ CTL and IFN-gamma in the control of RSV disease in infants and children is implicated.

Corrective effects of interleukin-12 on age-related deficiencies in IFN-gamma production and IL-12Rbeta2 expression in virus-specific CD8+ T cells.

Interleukin-12 receptor beta2 (IL-12Rbeta2) has been shown to be selectively expressed on Th1 T cell subsets, and we have previously shown that influenza-specific CD8+ cytotoxic T lymphocyte (CTL) deficiency in old mice was associated with deficient Th1 (interferon-gamma [IFN-gamma]) cytokine production. This study tested whether IL-12Rbeta2 expression was also deficient in CD8+ CTL from old mice and the effect of IL-12 treatment on these responses. Splenic lymphocytes from influenza-primed old and young BALB/c mice were stimulated with influenza virus in vitro with and without IL-12 and then enriched for CD8+ T cells. IFN-gamma was significantly reduced, whereas IL-4 and IL-12p40 (an antagonist of IL-12 function) were evaluated in old when compared with young mice. This was true for secreted protein measured by ELISA and for mRNA levels quantitated by RT-PCR. IL-12Rbeta2 mRNA expression in CD8+ CTL was also significantly reduced in old mice. IL-12 treatment in vitro caused significant upregulation of IFN-gamma and IL-12Rbeta2 and downregulation of IL-4 in CD8+ T cells from old mice and young mice. The present demonstration of an age-related downregulation in IL-12Rbeta2 expression and our previous data showing reduced IFN-gamma and elevated IL-4 production provide strong evidence that CD8+ CTL deficiency in aging results from a Th1/Th2 cytokine production switch. Agents that increase IL-12Rbeta2 expression and redirect Th2 to Thl immune responses are likely to enhance CD8+ CTL-mediated control of viral infections in aging.

Human interleukin-12 enhances interferon-gamma-producing influenza-specific memory CD8+ cytotoxic T lymphocytes.

Interferon (IFN)-gamma synthesis of CD45RO+ (memory) and CD45RA+ (naive) CD8+ cytotoxic T lymphocytes (CTLs) and the role of interleukin (IL)-12 in the regulation of human CTL functions in virus-specific immunity were investigated. After culture with influenza virus, CD45RO+ CD8+ T cells from human peripheral blood mononuclear cells increased in frequency and exhibited significant major histocompatibility complex class I-mediated CTL activity, whereas CD45RA+ CD8+ T cells did not. Influenza virus-stimulated CD45RO+ CD8+ T cells contained significantly higher levels of IFN-gamma-producing cells and IFN-gamma-specific mRNA than did CD45RA+ CD8+ T cells. Recombinant human IL-12 further enhanced CTL activity and IFN-gamma production by CD45RO+ CD8+ T cells. These data clearly show that human virus-specific CTL activity and coproduction of IFN-gamma are associated with the CD45RO+ CD8+ T cells that are modulated by the cell-mediated, immunity-inducible cytokine IL-12 in humans.

Mucosal immunity to influenza without IgA: an IgA knockout mouse model.

IgA knockout mice (IgA-/-) were generated by gene targeting and were used to determine the role of IgA in protection against mucosal infection by influenza and the value of immunization for preferential induction of secretory IgA. Aerosol challenge of naive IgA-/- mice and their wild-type IgA+/+ littermates with sublethal and lethal doses of influenza virus resulted in similar levels of pulmonary virus infection and mortality. Intranasal and i.p. immunization with influenza vaccine plus cholera toxin/cholera toxin B induced significant mucosal and serum influenza hemagglutinin-specific IgA Abs in IgA+/+ (but not IgA-/-) mice as well as IgG and IgM Abs in both IgA-/- and IgA+/+ mice; both exhibited similar levels of pulmonary and nasal virus replication and mortality following a lethal influenza virus challenge. Monoclonal anti-hemagglutinin IgG1, IgG2a, IgM, and polymeric IgA Abs were equally effective in preventing influenza virus infection in IgA-/- mice. These results indicate that IgA is not required for prevention of influenza virus infection and disease. Indeed, while mucosal immunization for selective induction of IgA against influenza may constitute a useful approach for control of influenza and other respiratory viral infections, strategies that stimulate other Igs in addition may be more desirable.

Targeted deletion of the IgA constant region in mice leads to IgA deficiency with alterations in expression of other Ig isotypes.

A murine model of IgA deficiency has been established by targeted deletion of the IgA switch and constant regions in embryonic stem cells. B cells from IgA-deficient mice were incapable of producing IgA in vitro in response to TGF-beta. IgA-deficient mice expressed higher levels of IgM and IgG in serum and gastrointestinal secretions and decreased levels of IgE in serum and pulmonary secretions. Expression of IgG subclasses was complex, with the most consistent finding being an increase in IgG2b and a decrease in IgG3 in serum and secretions. No detectable IgA Abs were observed following mucosal immunization against influenza; however, compared with those in wild-type mice, increased levels of IgM Abs were seen in both serum and secretions. Development of lymphoid tissues as well as T and B lymphocyte function appeared normal otherwise. Peyer's patches in IgA-deficient mice were well developed with prominent germinal centers despite the absence of IgA in these germinal centers or intestinal lamina propria. Lymphocytes from IgA-deficient mice responded to T and B cell mitogens comparable to those of wild-type mice, while T cells from IgA-deficient mice produced comparable levels of IFN-gamma and IL-4 mRNA and protein. In conclusion, mice with targeted deletion of the IgA switch and constant regions are completely deficient in IgA and exhibit altered expression of other Ig isotypes, notably IgM, IgG2b, IgG3, and IgE, but otherwise have normal lymphocyte development, proliferative responses, and cytokine production.

Cytokines and impaired CD8+ CTL activity among elderly persons and the enhancing effect of IL-12.

We have previously demonstrated that about 70% of elderly persons exhibit deficient cytotoxic T lymphocyte (CD8+ CTL) responses against influenza viruses when compared to young persons. Since IFN-gamma, a Th1 cytokine and IL-4, a Th2 cytokine, stimulate and inhibit CD8+ CTL responses respectively, their role(s) in the age-related CTL deficiency was investigated. Lymphocytes from young adults (34 +/- 5 years old) and elderly subjects (71 +/- 1 years old) were stimulated in vitro with influenza A/H3N2, A/H1N1 or influenza B virus for 6-7 days. The CD8+ CTL activity against virus-infected autologous target cells was significantly lower among the elderly than the young subjects (P < 0.01). Following stimulation with influenza virus, IL-4 production in both age groups was similar on day 3 but significantly higher among elderly persons on day 6 (P < 0.05). In contrast, T cells from the elderly produced significantly lower IFN-gamma than did those from young persons on both days (P < 0.05). Treatment of T cells from young and elderly adults with recombinant human IL-12, a pivotal cytokine that stimulates Th1 cytokines, resulted in enhancement of CD8+ CTL activity and IFN-gamma production in a dose dependent manner (P < 0.01). IL-12-dependent enhancement of CTL activity was not always abrogated by anti-IFN-gamma antibody treatment. These results suggest that deficient influenza virus-specific CTL activity among the elderly is attributable to a Th1 to Th2 cytokine production switch. Immunotherapy with IL-12 could represent a useful approach to correct the CD8+ CTL deficiency and cytokine imbalance among elderly humans.

Reversal of age-related deficient influenza virus-specific CTL responses and IFN-gamma production by monophosphoryl lipid A.

Old and young Balb/c mice, 24--26 and 2--4 months old, respectively, were infected with a 0.1 LD50 of influenza A/Taiwan/1/86 (A/H1N1) virus by small particle aerosol. Lung virus titers were determined 4, 6, 8, 12, and 17 days later. Old mice had significantly higher virus titers than young mice (P < 0.05-0.0001) and shed virus up to Day 17, while young mice were free of virus by Day 12. Splenic MHC class I CD8+ CTL activity (P < 0.08--0.001) and IFN-gamma production (0.1-0.008) measured on Days 8, 12, and 17 were significantly lower among old mice than among young mice. Coadministration of liposomal influenza vaccine with monophosphoryl lipid A (MPL) resulted in enhanced CD8+ CTL response and IFN-gamma production among old mice (35 and 12,000 times, respectively). These results demonstrate that MPL stimulates CTL and Th1 cytokines (IFN-gamma) in aged mice and may serve to reverse age-related CD8+ CTL deficiency and reduce severe influenza disease in elderly human populations.

Cytotoxic T lymphocyte responses of infants after natural infection or immunization with live cold-recombinant or inactivated influenza A virus vaccine.

The cytotoxic T lymphocyte (CTL) response of infants after immunization with either inactivated trivalent subvirion vaccine (TIV) or bivalent attenuated cold-recombinant (CR) vaccine or occurrence of natural influenza virus infection were compared in a blinded, placebo-controlled study during the 1987-1988 and 1988-1989 influenza epidemic seasons. Healthy infants between 6 and 13 months of age were randomly assigned and administered a single dose of intranasal bivalent (A/H3N2/A/H1N1) CR vaccine, a two-dose regimen of TIV (A/H3N2/A/H1N1/B) influenza vaccine, or placebo. Peripheral blood lymphocytes were obtained prior to and 2-8 weeks after vaccination and at the end of the epidemic season and stimulated with virus in vitro for 6 or 7 days. Lysis of autologous virus-infected target cells was assessed in a 4 hr 51Cr release assay. MHC class I-restricted influenza A-specific CTL was stimulated following natural influenza A virus infection but not after immunization with CR influenza A virus vaccine or TIV. These results demonstrate for the first time induction of influenza virus-specific CTL activity in infants under 1 year of age.

Influenza A subtype cross-protection after immunization of outbred mice with a purified chimeric NS1/HA2 influenza virus protein.

Influenza A/PR/8/34-derived chimeric (D) protein (SK&F 106160) composed of the first 81 amino acids (aa) of NS1 fused to the conserved 157 C-terminal aa of HA2 (NS1 1-81-HA2 65-222) was previously shown to induce H-2d-restricted protective cytotoxic T-lymphocyte (CTL) immunity in inbred mice. However, D protein, like other small peptides, exhibited haplotype dependence and was not immunogenic in H-2b and H-2K mice. A potential use of this antigen in humans and the role of T cells in any protection were evaluated in outbred Swiss and inbred CBF6F1 (H-2d/b) mice. Mice immunized with D protein and challenged by small-particle aerosol with a lethal dose of influenza virus were significantly protected against mortality from influenza A/H1N1 and A/H2N2 (p < 0.05-< 0.0000001), but not from A/H3N2 and influenza B viruses when compared with control mice. D protein did not induce serum virus-neutralizing antibody but caused virus to be cleared faster in immunized mice. Protection was long-lasting. In vivo depletion of either Lyt2 (CD8+) or L3T4 (CD4+) T cells with monoclonal antibodies led to abrogation of in vitro-generated CTL activity in CF6F1 mice and significant reduction in the protective efficacy of D protein against virus challenge in both Swiss and CF6F1 mice. These results suggest that protection was mediated by CD8+ and/or CD4+ cells and not antibody. Thus D protein, via a conserved sequence on the HA2 polypeptide, has the potential to induce partially cross-reactive CTL that may protect against influenza virus disease in humans.

Quantitation of human influenza virus-specific cytotoxic T lymphocytes: correlation of cytotoxicity and increased numbers of IFN-gamma producing CD8+ T cells.

To study virus-specific cytotoxic T lymphocyte (CTL) activity at the single cell level, an IFN-gamma specific ELISPOT assay was adapted to elucidate the frequency of influenza-specific CTLs together with a standard cytotoxic 51Cr-release assay. Peripheral blood mononuclear cells (PBMCs) from human volunteers were cultured with influenza virus-infected autologous cells; following 3 or 7 days of culture, T cell subsets were assessed for IFN-gamma production by IFN-gamma-specific ELISPOT and ELISA, while IFN-gamma mRNA expression was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). Influenza virus-specific CTL activity was measured in a 4 h 51Cr-release assay. Culture of PBMC with autologous A/Taiwan influenza (H1N1)-infected stimulator cells resulted in IFN-gamma spot forming cells (SFCs) at 3 days that increased after 7 days of incubation. Numbers of IFN-gamma SFCs directly correlated with levels of secreted IFN-gamma and higher levels were seen in supernatants from 7 day cultures. RT-PCR analysis (35 cycles of amplification) showed greater IFN-gamma mRNA in T cells isolated from 7 day cultures. Separate aliquots of T cells from these cultures were also assessed for virus-specific cytotoxicity and T cells from 7 day (but not from 3 day) cultures induced high 51Cr release. Analysis indicated a significant direct correlation between level of cytotoxicity, number of IFN-gamma SFCs, and amount of IFN-gamma in culture supernatants. Studies with purified T cell subsets showed that elevated IFN-gamma SFCs, IFN-gamma synthesis, and cytotoxic activity were associated with CD4-CD8+ T cells but not with the CD4+CD8- T cell subset.(ABSTRACT TRUNCATED AT 250 WORDS)

Maternal immunization with influenza or tetanus toxoid vaccine for passive antibody protection in young infants.

Women in the last trimester of pregnancy were given trivalent inactivated influenza virus vaccine (TIV; A/Sichuan/H3N2, A/Taiwan/H1N1, B/Victoria) or tetanus toxoid (TT). Maternal blood was drawn before immunization and at delivery (median, 5 weeks later); infant blood was obtained within 5 days of birth and 2 months later. Antibody responses to TIV and TT were determined by microneutralization assay and ELISA. T cell response was determined by lymphocyte proliferation. Maternal seroconversion to vaccine antigens was found to one or more influenza antigen in all TIV recipients and to TT in 9 of 13 TT recipients. Significantly higher IgG antibodies to maternal vaccine antigens were present in cord and infant serum. Significant blastogenic responses were seen to influenza A and B in maternal cells of TIV-immunized women but not in cord or infant lymphocytes. Maternal immunization resulted in higher infant levels of vaccine-specific IgG antibody but not in the transfer of specific T lymphocyte response(s) or production of neonatal IgM antibody.

Induction of CD8+ cytotoxic T cells by immunization with killed influenza virus and effect of cholera toxin B subunit.

The MHC class I cytotoxic T-lymphocyte (CTL) response in mice given formalin-inactivated influenza whole-virus vaccine (WVV) with or without cholera toxin B subunit (CTB) was studied. Intraperitoneal injection of Balb/c (H-2d) mice with high doses of A/Taiwan/1/86 (H1N1) WVV stimulated influenza A virus-specific CTL response in a dose-dependent manner. A dose of 4.4 or 44 micrograms induced CTL response equal to or greater than live influenza virus infection. Coadministration of vaccine with 5 or 25 micrograms of CTB resulted in a higher level of CTL than with vaccine alone. CTL lysed A/Taiwan and A/Shanghai (H3N2) virus-infected class I-expressing P815 (H-2d) but not virus-infected EL-4 (H-2b) target cells nor B/Yamagata virus-infected target cells. Virus-infected MHC class II- and class I-expressing A20 (H-2d) targets were also lysed. Depletion of Lyt-2+ (CD8+) T cells with monoclonal antibody completely abrogated lysis of P815 target cells and resulted only in a slight reduction of lysis of A20 target cells. Depletion of L3T4+ (CD4+) T cells or NK cells had minimal effect on lysis of either P815 or A20 target cells. Using limiting dilution analysis, the precursor CTL (pCTL) frequency paralleled CTL activity. Significant CTL activity was detected 7 months after immunization. These results demonstrate that adequate doses of influenza WVV with or without CTB can induce long-lasting influenza A cross-reactive MHC class I-restricted CD8+ CTL response in mice. Thus, coadministration of influenza WVV with CTB may lead to an effective vaccine that stimulates both CTL and antibody responses.

Studies on reactogenicity and immunogenicity of attenuated bivalent cold recombinant influenza type A (CRA) and inactivated trivalent influenza virus (TI) vaccines in infants and young children.

Fifty-two infants seronegative to or without prior infection with influenza type A viruses were enrolled in a study to evaluate reactogenicity and immunogenicity of three bivalent cold recombinant type A (CRA) and two trivalent inactivated influenza (TI) vaccines. Controls consisted of infants receiving normal saline by nose drops (Pli.n.) or intramuscularly (Pli.m.). CRA and TI vaccines were monitored for local and systemic reactions after vaccination. Serum specimens obtained prior to and 6 weeks postvaccination were analysed for neutralizing antibody to influenza H1N1 and H3N2 viruses. CRA vaccines and Pli.n. recipients had similar numbers of acute respiratory infections and comparable rates of illnesses during the trial. Significantly fewer CRA vaccinees without an intercurrent viral infection had fever (0/16 versus 4/10, p = 0.04) and cough (4/16 versus 9/10, p = 0.002) than CRA vaccinees with a confirmed intercurrent viral infection. Recipients of TI vaccine and Pli.m. did not develop reactions at the injection site. For each of the CRA vaccines tested, a dominant CRA virus was identified. The dominant CRA viruses were isolated from a greater number of infants or for a longer duration than the non-dominant CRA viruses. All 14 non-dominant CRA viruses were recovered from infants within the first week after vaccination; 24 of 77 dominant CRA viruses were recovered more than 7 days after vaccination. The immunogenicity of CRA vaccines was not affected by a confirmed intercurrent viral infection or low titres of influenza-specific antibody.(ABSTRACT TRUNCATED AT 250 WORDS)

Diminished influenza A virus-specific MHC class I-restricted cytotoxic T lymphocyte activity among elderly persons.

Influenza A virus-specific MHC class I-restricted cytotoxic T lymphocyte (CTL) activities among young and elderly adults were compared. Peripheral blood lymphocytes from 10 young adults, (mean age 27 +/- 2.4 years) and elderly persons (mean age 71 +/- 1.6 years) were stimulated with influenza A/Taiwan/1/86 (H1N1) virus for 7 days and assayed for lytic activity against A/Taiwan, A/Shanghai (H3N2), and B/USSR virus-infected autologous target cells. Young adults exhibited significantly higher influenza A cross-reactive CTL activity against A/H1N1 and A/H3N2 target cells when compared to aged persons. This was true at all effector-to-target cell ratios tested. Negligible lysis of B/USSR-infected target cells or nonautologous A/Taiwan-infected cells was observed. The number of leukocytes recovered per milliliter of blood was also significantly higher in young adults than in old donors; however, the percentage of CD45+ (common leukocyte antigen), CD3+ (T cells), CD4+ (T helper), and CD8+ (T cytotoxic/suppressor) as well as the CD4+/CD8+ ratios was similar in both groups. Depletion of cells with monoclonal antibodies indicated that the effector cells were CD8+ T cells. Serum-neutralizing antibody (Nt Ab) titers were similar among young and elderly persons and there was no correlation between Nt Ab and CTL activity. These results demonstrate a reduced influenza virus-specific MHC class I-restricted CTL activity among elderly persons. The deficiency in this cell-mediated immune function may contribute to the morbidity and mortality from influenza virus infections in this population.

Enhancement of the protective efficacy of inactivated influenza A virus vaccine in aged mice by IL-2 liposomes.

A dose-dependent, vaccine-induced protection of aged and young Balb/c mice against lethal influenza A virus challenge has been demonstrated. Low dose formalin-inactivated influenza A virus vaccine was protective in young mice, but not in aged mice, while a higher dose was protective in both. Administration of low dose vaccine with IL-2 liposomes conferred protection comparable to the high dose in aged mice. Serum neutralizing antibody responses were stimulated by vaccine in a dose-dependent manner while IL-2 liposomes significantly enhanced responses in the low dose paralleled protection in young but not in aged mice. Lung interferon levels paralleled lung virus titres in young but not in aged mice. CTL responses in infected mice were generally higher in young than aged mice. These results demonstrate efficacy of IL-2 liposomes as an adjuvant for influenza virus vaccines in the aged.

Vaccination with inactivated influenza A virus during pregnancy protects neonatal mice against lethal challenge by influenza A viruses representing three subtypes.

A single intraperitoneal injection of pregnant mice with a monovalent Formalin-inactivated influenza A virus vaccine protected their offspring against a lethal challenge dose of the same influenza A virus H3N2, H2N2, and H1N1 subtypes, as well as against challenge with the other two subtypes. Degree of protection was vaccine dose related. Cross-fostering of neonates indicated that protection was conferred by breast milk antibodies. Serum virus-specific neutralizing antibodies in the mothers and neonates correlated with resistance to vaccine virus, but were detected against other subtypes only in a complement enhancement test or when high doses of vaccine were given.