Zichria Zakay-Rones

High Seroprevalence of Human Metapneumovirus among Young Children in Israel

Exposure to the newly discovered human metapneumovirus (HMPV) during the first 2 years of life was studied by longitudinal serological analysis in 40 healthy children in southern Israel. The seropositivity rate decreased to a minimum by age 13 months and increased to 52% by age 24 months. Evidence of new infection was detected in 13%, 23%, and 55% of children by ages 7, 13, and 24 months, respectively. The high exposure rates suggest that HMPV may be an important cause of community-acquired respiratory-tract infections in young children.

Liposomal immunostimulatory DNA sequence (ISS-ODN): an efficient parenteral and mucosal adjuvant for influenza and hepatitis B vaccines.

Synthetic oligodeoxynucleotides (ODNs) containing immunostimulatory sequences (ISS-ODN, also known as CpG-ODNs) have been shown to display in experimental models potent Th1-biassed immunoadjuvant activity upon parenteral or mucosal co-administration with a variety of antigens. In an attempt to potentiate adjuvant activity, and to reduce dose and number of administrations, ISS-ODN was entrapped (up to 90% efficiency) in large (1.5 microm) multilamellar liposomes using a simple and fast (5 min) procedure. Mice were vaccinated once or twice intramuscularly (i.m.) or intranasally (i.n.) with subunit influenza vaccines (consisting of the viral hemagglutinin and neuraminidase, HN) or with hepatitis B surface antigen particles (HBsAg), either non-encapsulated or liposome-encapsulated, together with free or liposomal ISS-ODN (5-25 microg per dose). At 3-12 weeks post-vaccination, the humoral (systemic, mucosal) and cellular responses and protective immunity were assessed. Vaccine formulations containing liposomal ISS-ODN co-administered with either soluble antigen or liposomal antigen (in the same vesicles or in separate vesicles) were up to 30 times more effective than formulations containing un-encapsulated ISS-ODN in inducing: (a) antigen-specific serum and mucosal IgG2a and IgA antibodies; (b) splenocyte proliferative response, cytotoxic activity and IFNgamma production; (c) a DTH response; and (d) protection against virus challenge. The response was Th1-dominant in the influenza model and a mixed Th1+Th2 response in the hepatitis B model. No adverse reactions were noted. Thus, liposomal encapsulation of ISS-ODN further enhances its inherent adjuvant activity.

Dehydroepiandrosterone (DHEA) treatment reverses the impaired immune response of old mice to influenza vaccination and protects from influenza infection

Dehydroepiandrosterone (DHEA) is a native steroid with an immunomodulating activity. Recently it was suggested that its age-associated decline is related with immunosenescence. To examine whether DHEA administration could effectively reverse the age-associated decline of immunity against influenza vaccine, aged mice were simultaneously vaccinated and treated with DHEA. Reversal of the age-associated decline and a significant constant increase of humoral response was observed in treated mice. Increased resistance to post-vaccination intranasal challenge with live influenza virus was observed in DHEA-treated aged mice. Thus, DHEA treatment overcame the age-related defect in the immunity of old mice against influenza.

A novel liposomal influenza vaccine (INFLUSOME-VAC) containing hemagglutinin–neuraminidase and IL-2 or GM-CSF induces protective anti-neuraminidase antibodies cross-reacting with a wide spectrum of influenza A viral strains

A liposomal influenza vaccine (INFLUSOME-VAC) was developed with the objective of overcoming the major drawbacks of the currently used influenza vaccines: their relatively low efficacy in certain high-risk groups (the elderly, infants, the immunosuppressed) and the need for annual immunization. INFLUSOME-VAC consists of liposomes containing the viral surface proteins hemagglutinin (HA) and neuraminidase (NA) derived from various influenza strains and IL-2 or GM-CSF, as an adjuvant. Vaccination of mice showed that, whereas conventional vaccines induced a low- and short-term response against HA and very low or no anti-NA response, INFLUSOME-VAC produced high titers of both anti-HA and anti-NA antibodies (Abs) in young and old mice that persisted for at least 6 months. Moreover, the anti-NA Abs efficiently cross-reacted with several N2 viral subtypes spanning 20 years, and such vaccines afforded partial protection against heterosubtypic viral infection.

Antibody response to influenza immunization in patients after heart transplantation

The aim of this study was to evaluate post-heart transplantation (Htx) response to two-dose and three-dose influenza vaccine. Hemagglutination inhibition antibodies were monitored in HTx recipients immunized twice (n = 25) or three times (n = 17), and non-HTx controls (n = 8) once, with inactivated influenza vaccine. Post-first dose protective antibody titers (> or = 1:40) were demonstrated in 9/25 (36%) for A/Singapore/ (H1N1), 5/25 (20%) for A/Shanghai/(H3N2) and 2/25 (8%) for B/Yamagata compared with 4/8 (50%), 6/8 (75%) and 2/8 (25%), respectively, for controls. Post-second dose protective titers remained low, increasing following the third dose to 71%, 65% and 29%, respectively. The abnormally low antibody responses of HTx recipients to one-dose and two-dose influenza vaccine can be overcome by a third dose.

A novel influenza subunit vaccine composed of liposome-encapsulated haemagglutinin/neuraminidase and IL-2 or GM-CSF. I. Vaccine characterization and efficacy studies in mice

The aim of this study was to improve the potency of the currently used influenza subunit vaccines, which are of relatively low efficiency in high-risk groups. Influenza A virus (Shangdong/9/93) haemagglutinin/neuraminidase (H3N2), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2) were encapsulated, each separately or combined, in multilamellar vesicles composed of dimyristoyl phosphatidylcholine. BALB/c mice were immunized once, i.p. or s.c., with 0.05-2.0 microg HN administered either as free antigen (F-HN), adsorbed to aluminum hydroxide (Al-HN), or encapsulated in liposomes (Lip-HN), separately or together with 1 x 10(2)-4.5 x 10(4) units of free or encapsulated cytokines. Serum antibodies were assayed on days 11-360 by the haemagglutination-inhibition (HI) test and ELISA. Protective immunity against intranasal virus challenge was determined at 9-14 months post-vaccination. The following results were obtained: (1) The efficiency of encapsulation in liposomes was 95, 90 and 38% for HN, IL-2 and GM-CSF, respectively, and the liposomal preparations were highly stable as an aqueous dispersion for > 2 months at 4 degrees C. (2) Following immunization with 0.5 microg Lip-HN, there was an earlier, up to 50-fold stronger, and 3-5 times longer response than that obtained with nonliposomal HN. (3) Coimmunization with free cytokines further increased the response 2-20 times and the two cytokines had an additive effect. (4) Liposomal cytokines were 2-20 times more effective than the free cytokines and their stimulatory effect was more durable. (5) A 100% seroconversion (HI titer > or = 40) was achieved with only 10-25% of the routinely used antigen dose, by encapsulating either antigen or cytokine. (6) The level of protection following vaccination with the combined liposomal vaccines was 70-100% versus 0-25% in mice immunized with Al-HN alone, and no toxicity was observed. In conclusion, our animal experiments show that the liposomal vaccines are superior to the currently used influenza vaccines, increasing the response by 2-3 orders of magnitude in mice. This approach may also prove valuable for subunit vaccines against other microorganisms.

Immunogenicity and safety of a novel IL-2-supplemented liposomal influenza vaccine (INFLUSOME-VAC) in nursing-home residents.

Influenza and its complications account for substantial morbidity and mortality, especially among the elderly. In young adults, immunization provides 70-90% protection, while among the elderly the vaccine may be only </=50% effective; hence, the need for new, more immunogenic vaccines. We compared the safety and immunogenicity of a novel, interleukin-2 (IL-2) -supplemented trivalent liposomal influenza vaccine (designated INFLUSOME-VAC) with that of a commercial trivalent split virion vaccine in community-residing elderly volunteers (mean age 81 years) in winter of 2000/2001. Eighty-one individuals were randomly assigned to be vaccinated intramuscularly, either with the standard vaccine (n=33) or with INFLUSOME-VAC (n=48) prepared from the former. The two vaccines contained equal amounts of hemagglutinin (HA) ( approximately 15 microgram of each viral strain); INFLUSOME-VAC consisted of liposomal antigens admixed with liposomal human IL-2 (Lip IL-2) (33 microgram = 6x10(5) IU/dose). At 1 month post-vaccination, seroconversion rates (tested by hemagglutination inhibition) for the A/New Caledonia (H1N1) and A/Moscow (H3N2) strains were significantly higher (P=0.04) in the INFLUSOME-VAC group (65 versus 45%, 44 versus 24%, respectively). Moreover, INFLUSOME-VAC induced a greater anti-neuraminidase (NA-N2) response (P<0.05). Anti-IL-2 antibodies were undetected, and no increase in anti-phospholipid IgG antibodies was found in the INFLUSOME-VAC group. Adverse reactions were similar in both groups. Thus, INFLUSOME-VAC appears to be both safe and more immunogenic than the currently used vaccine in the elderly.

Local and systemic immune response in community-dwelling elderly after intranasal or intramuscular immunization with inactivated influenza vaccine.

Intramuscular (IM) influenza vaccines are about 50% effective in preventing clinical illness among the elderly and their effectiveness in eliciting mucosal response may be even lower. The aim of the present study was to evaluate the immunological effect of a novel inactivated intranasal (IN) trivalent whole influenza virus vaccine among community‐dwelling elderly. Sixty‐one subjects were vaccinated with two doses of an IN vaccine and a control group of 31 subjects was vaccinated with a commercial IM vaccine. Viral strains in the 1997/8 vaccine used were A/Nanchang/933/95(H3N2), A/Johannesburg/82/96(H1N1) and B/Harbin/7/94. Serum IgG and nasal IgA were determined by HI and ELISA, respectively. Only a few minor local adverse events were reported after vaccination. Seroconversion for the three antigens tested was higher after IM vaccination, although not statistically significant. Local antibody response to the three antigens tested was detected in 50–53% and 19–26% of IN and IM immunized subjects, respectively. The IN vaccine tested was significantly more effective than the IM vaccine in inducing mucosal IgA response. This may prevent influenza at its early stages and thus contribute to the reduction of complications in the elderly. J. Med. Virol. 61:100–106, 2000.

The Oncolytic Activity of Newcastle Disease Virus NDV-HUJ on Chemoresistant Primary Melanoma Cells Is Dependent on the Proapoptotic Activity of the Inhibitor of Apoptosis Protein Livin

ABSTRACT Patients with advanced melanoma usually do not benefit from conventional chemotherapy treatment. There is therefore a true need for a new kind of therapy for melanoma. One factor responsible for the poor prognosis of melanoma is the inhibitor of apoptosis protein (IAP) family member Livin. In this study, we applied a novel approach for the treatment of melanoma, using a unique strain of the oncolytic Newcastle disease virus (NDV-HUJ). We found that, unlike chemotherapeutic drugs, NDV-HUJ, a one-cycle replicating virus, overcomes the resistance to apoptosis of melanoma primary cultures that over express the Livin protein. In contrast, melanoma tumor cells that do not express Livin are relatively resistant to NDV-HUJ treatment. Furthermore, we show that NDV-HUJ-induced oncolysis is attributed to the dual function of Livin: although Livin inhibits apoptosis through the inhibition of caspases, under the robust apoptotic stimulation of NDV-HUJ, caspases can cleave Livin to create a truncated protein with a paradoxical proapoptotic activity. Thus, NDV-HUJ is a potent inducer of apoptosis that can overcome the antiapoptotic effect of Livin and allow cleavage of Livin into the proapoptotic tLivin protein. Moreover, the results indicate that the interferon system, which is functional in melanoma, is not involved in NDV-induced oncolysis. Taken together, our data offer the possibility of a new viral oncolytic treatment for chemoresistant melanoma.

A novel influenza subunit vaccine composed of liposome-encapsulated haemagglutinin/neuraminidase and IL-2 or GM-CSF. II. Induction of TH1 and TH2 responses in mice

This study was aimed at analyzing, in parallel, the humoral and cellular immune responses elicited in mice immunized with liposomal influenza A (Shangdong/9/93) subunit vaccines composed of haemagglutinin/neuraminidase (H3N2) and IL-2 or GM-CSF. Recently, we reported that such vaccines evoke a more rapid, stronger and longer-lasting (over 1 year) humoral response, as well as protective immunity against viral infection, following a single administration, as compared with the response induced by the free antigen given alone or together with soluble cytokines. In the present study, BALB/C mice were immunized once, i.p., s.c., i.m. or i.n., with nonliposomal or liposomal vaccines and the humoral (antibody titer and isotypes) and cellular (DTH, cytotoxicity, cytokine production) responses were assessed at various times (2-56 weeks). The main findings were: (a) the combined liposomal vaccines consisting of encapsulated antigen and encapsulated cytokine, but not the free antigen, elicited a high titer of serum IgG1, IgG2a, IgG3 and IgM antibodies; (b) the combined liposomal vaccines were efficient following administration by the various routes, and induced a local (in lung) IgA response in i.n. vaccinated mice; (c) the liposomal vaccines triggered DTH and cytotoxic responses, as well as cytokine (mainly IL-4) production. Together, these and other findings indicate that our cytokine-supported liposomal influenza vaccines efficiently stimulate both Th1 and Th2 responses and that such vaccines may be more potent in high-risk groups than the currently used subunit vaccines.