RESUMEN
Herpes simplex virus type 2 (HSV-2) is the most common cause of genital disease worldwide. The development of an effective HSV-2 vaccine would significantly impact global health based on the psychological distress caused by genital herpes for some individuals, the risk transmitting the infection from mother to infant, and the elevated risk of acquiring HIV-1. Five nonclinical safety studies were conducted with the replication defective HSV529 vaccine, alone or adjuvanted with GLA-SE, and the G103 subunit vaccine containing GLA-SE. A biodistribution study was conducted in guinea pigs to evaluate distribution, persistence, and shedding of HSV529. A preliminary immunogenicity study was conducted in rabbits to demonstrate HSV529-specific humoral response and its enhancement by GLA-SE. Three repeated-dose toxicity studies, one in guinea pigs and two in rabbits, were conducted to assess systemic toxicity and local tolerance of HSV529, alone or adjuvanted with GLA-SE, or G103 containing GLA-SE. Data from these studies show that both vaccines are safe and well tolerated and support the ongoing HSV-2 clinical trial in which the two vaccine candidates will be given either sequentially or concomitantly to explore their potential synergistic and incremental effects.
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Anticuerpos Antivirales , Herpesvirus Humano 2 , Humanos , Animales , Cobayas , Conejos , Distribución Tisular , Proteínas del Envoltorio Viral , Adyuvantes Inmunológicos , Vacunas de SubunidadRESUMEN
mRNA vaccines hold tremendous potential in disease control and prevention for their flexibility with respect to production, application, and design. Recent breakthroughs in mRNA vaccination would have not been possible without major advances in lipid nanoparticles (LNPs) technologies. We developed an LNP containing a novel ionizable cationic lipid, Lipid-1, and three well known excipients. An in silico toxicity hazard assessment for genotoxicity, a genotoxicity assessment, and a dose range finding toxicity study were performed to characterize the safety profile of Lipid-1. The in silico toxicity hazard assessment, utilizing two prediction systems DEREK and Leadscope, did not find any structural alert for mutagenicity and clastogenicity, and prediction in the statistical models were all negative. In addition, applying a read-across approach a structurally very similar compound was tested negative in two in vitro assays confirming the low genotoxicity potential of Lipid-1. A dose range finding toxicity study in rabbits, receiving a single intramuscular injection of either different doses of an mRNA encoding Influenza Hemagglutinin H3 antigen encapsulated in the LNP containing Lipid-1 or the empty LNP, evaluated local tolerance and systemic toxicity during a 2-week observation period. Only rabbits exposed to the vaccine were able to develop a specific IgG response, indicating an appropriate vaccine take. The vaccine was well tolerated up to 250 µg mRNA/injection, which was defined as the No Observed Adverse Effect Level (NOAEL). These results support the use of the LNP containing Lipid-1 as an mRNA delivery system for different vaccine formulations and its deployment into clinical trials.
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Lípidos , Nanopartículas , Animales , Lípidos/química , Lípidos/toxicidad , Liposomas , Nanopartículas/química , Nanopartículas/toxicidad , ARN Mensajero/genética , ConejosRESUMEN
The use of modified nucleosides is an important approach to mitigate the intrinsic immunostimulatory activity of exogenous mRNA and to increase its translation for mRNA therapeutic applications. However, for vaccine applications, the intrinsic immunostimulatory nature of unmodified mRNA could help induce productive immunity. Additionally, the ionizable lipid nanoparticles (LNPs) used to deliver mRNA vaccines can possess immunostimulatory properties that may influence the impact of nucleoside modification. Here we show that uridine replacement with N1-methylpseudouridine in an mRNA vaccine encoding influenza hemagglutinin had a significant impact on the induction of innate chemokines/cytokines and a positive impact on the induction of functional antibody titers in mice and macaques when MC3 or KC2 LNPs were used as delivery systems, while it impacted only minimally the titers obtained with L319 LNPs, indicating that the impact of nucleoside modification on mRNA vaccine efficacy varies with LNP composition. In line with previous observations, we noticed an inverse correlation between the induction of high innate IFN-α titers in the macaques and antigen-specific immune responses. Furthermore, and consistent with the species specificity of pathogen recognition receptors, we found that the effect of uridine replacement did not strictly translate from mice to non-human primates.
RESUMEN
The World Health Organization protocol for rabies post-exposure prophylaxis (PEP) recommends extensive wound washing, immediate vaccination, and administration of rabies immunoglobulin (RIG) in severe category III exposures. Some studies have shown that RIG can interfere with rabies vaccine immunogenicity to some extent. We investigated the interference of RIG on a next generation highly purified Vero cell rabies vaccine candidate (PVRV-NG) versus standard-of-care vaccines in a previously described hamster model. The interference of either human (h) or equine (e) RIG on the immune response elicited by PVRV-NG, Verorab® (purified Vero cell rabies vaccine, PVRV), and Imovax® Rabies (human diploid cell rabies vaccine; HDCV) was evaluated using the 4-dose Essen PEP regimen. The anti-rabies seroneutralizing titers and specific serum IgM titers were measured by fluorescent antibody virus neutralization test and enzyme-linked immunosorbent assay, respectively, for the vaccines administered with or without RIG. The RIG interference on PVRV-NG, observed transiently at Day 7, was similar to that on PVRV and tended to be lower than that on HDCV using both read-outs. In summary, the results generated in the hamster model showed that RIG induced similar or less interference on PVRV-NG than the standard-of-care vaccines.
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Antígenos de Grupos Sanguíneos , Vacunas Antirrábicas , Virus de la Rabia , Rabia , Animales , Anticuerpos Antivirales , Chlorocebus aethiops , Cricetinae , Caballos , Humanos , Inmunoglobulinas , Factores Inmunológicos , Profilaxis Posexposición , Rabia/prevención & control , Vacunas Antirrábicas/inmunología , Virus de la Rabia/inmunología , Células VeroRESUMEN
The mRNA vaccine technology has promising applications to fight infectious diseases as demonstrated by the licensing of two mRNA-based vaccines, Comirnaty® (Pfizer/BioNtech) and Spikevax® (Moderna), in the context of the Covid-19 crisis. Safe and effective delivery systems are essential to the performance of these vaccines and lipid nanoparticles (LNPs) able to entrap, protect and deliver the mRNA in vivo are considered by many as the current "best in class". Nevertheless, current mRNA/LNP vaccine technology has still some limitations, one of them being thermostability, as evidenced by the ultracold distribution chain required for the licensed vaccines. We found that the thermostability of mRNA/LNP, could be improved by a novel imidazole modified lipid, DOG-IM4, in combination with standard helper lipids. DOG-IM4 comprises an ionizable head group consisting of imidazole, a dioleoyl lipid tail and a short flexible polyoxyethylene spacer between the head and tail. Here we describe the synthesis of DOG-IM4 and show that DOG-IM4 LNPs confer strong immunization properties to influenza HA mRNA in mice and macaques and a remarkable stability to the encapsulated mRNA when stored liquid in phosphate buffered saline at 4 °C. We speculate the increased stability to result from some specific attributes of the lipid's imidazole head group.
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COVID-19 , Nanopartículas , Animales , COVID-19/prevención & control , Imidazoles , Inmunización , Lípidos , Liposomas , Ratones , Primates/genética , ARN Mensajero/genética , Vacunas Sintéticas , Vacunas de ARNmRESUMEN
Current vaccination strategies against influenza focus on generating an antibody response against the viral haemagglutination surface protein, however there is increasing interest in neuraminidase (NA) as a target for vaccine development. A critical tool for development of vaccines that target NA or include an NA component is available validated serology assays for quantifying anti-NA antibodies. Additionally serology assays have a critical role in defining correlates of protection in vaccine development and licensure. Standardisation of these assays is important for consistent and accurate results. In this study we first validated a harmonized enzyme-linked lectin assay (ELLA)- Neuraminidase Inhibition (NI) SOP for N1 influenza antigen and demonstrated the assay was precise, linear, specific and robust within classical acceptance criteria for neutralization assays for vaccine testing. Secondly we tested this SOP with NA from influenza B viruses and showed the assay performed consistently with both influenza A and B antigens. Third, we demonstrated that recombinant NA (rNA) could be used as a source of antigen in ELLA-NI. In addition to validating a harmonized SOP we finally demonstrated a clear improvement in inter-laboratory agreement across several studies by using a calibrator. Importantly we showed that the use of a calibrator significantly improved agreement when using different sources of antigen in ELLA-NI, namely reverse genetics viruses and recombinant NA. We provide a freely available and detailed harmonized SOP for ELLA-NI. Our results add to the growing body of evidence in support of developing biological standards for influenza serology.
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Vacunas contra la Influenza , Gripe Humana , Anticuerpos Antivirales , Humanos , Lectinas/metabolismo , Neuraminidasa/genética , Reproducibilidad de los Resultados , Genética InversaRESUMEN
PURPOSE: The objective of this study was to investigate the potential of rapidly gelling nasal inserts as vaccine delivery system. METHODS: Nasal inserts were prepared by freeze-drying hydrophilic polymer solutions containing influenza split vaccine. In vitro vaccine release from polymer solutions and inserts and the vaccine hemagglutination activity were determined. In vivo immunization studies in mice and rats were performed with nasal solutions and nasal inserts. RESULTS: The in vitro release of proteins (vaccine) from polymeric solutions and inserts was incomplete because of the high molecular weight of the proteins. The release rate was controlled by the polymer (Lutrol F68 > PVP 90 > HPMC K15M > Carbopol > chitosan > or = carrageenan = xanthan gum) because of differences in solution viscosity and possible polymer-protein interactions. Xanthan gum, a negatively charged polymer with intrinsic adjuvanticity, enhanced the serum IgG as well as the nasal IgA response in in vivo studies with nasal vaccine solutions. Poly-l-arginine and cationic lipid were the best performing adjuvants. Solutions containing vaccine with xanthan gum and cationic lipid were effectively stabilized with 0.4 M NaCl. DISCUSSION: The specific activity of the major vaccine protein, hemagglutinin, was not significantly affected by the addition of polymers and the freeze-drying process during insert preparation. The addition of cationic lipid as adjuvant decreased the hemagglutination activity, which strongly indicated inhibition of the protein binding site to erythrocytes. Inserts prepared from xanthan gum and cationic lipid stabilized with NaCl showed a reduced protein activity but were superior to the cationic lipid alone. CONCLUSION: Rat immunization with solid nasal inserts based on xanthan gum containing the influenza vaccine, with or without an additional cationic lipid adjuvant, resulted in similar IgG levels as the pure nasal liquid vaccine formulation.
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Portadores de Fármacos/administración & dosificación , Subtipo H1N1 del Virus de la Influenza A , Vacunas contra la Influenza/administración & dosificación , Administración Intranasal , Animales , Portadores de Fármacos/farmacocinética , Sistemas de Liberación de Medicamentos , Femenino , Geles , Hemaglutininas Virales/administración & dosificación , Hemaglutininas Virales/metabolismo , Subtipo H1N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/farmacocinética , Ratones , Polisacáridos Bacterianos/administración & dosificación , Polisacáridos Bacterianos/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Hemagglutinin, the major surface protein of influenza viruses, was recombinantly expressed in eukaryotic cells as a monomer instead of its native trimer, and was only immunogenic when administered with an adjuvant [Pion et al. 2014]. In order to multimerize this antigen to increase its immunogenicity, a cysteine-rich peptide sequence found at the extreme C-terminus of lamprey variable lymphocyte receptor (VLR)-B antibodies was fused to various recombinant hemagglutinin (rHA) proteins from A and B influenza virus strains. The rHA-Lamp fusion (rHA fused to the lamprey sequence) protein was expressed in Leishmania tarentolae and Chinese hamster ovary (CHO) cells and shown to produce several multimeric forms. The multimers produced were very stable and more immunogenic in mice than monomeric rHA. The lamprey VLR-B sequence was also used to multimerize the neuraminidase (NA) of influenza viruses expressed in CHO cells. For some viral strains, the NA was expressed as a tetramer like the native viral NA form. In addition, the lamprey VLR-B sequence was fused with two surface antigens of Shigella flexneri 2a, the invasion plasmid antigen D and a double mutated soluble form of the membrane expression of the invasion plasmid antigen H namely MxiH. The fusion proteins were expressed in Escherichia coli to produce the respective multimer protein forms. The resulting proteins had similar multimeric forms as rHA-Lamp protein and were more immunogenic in mice than the monomer forms. In conclusion, the VLR-B sequence can be used to increase the immunogenicity of recombinant viral and bacterial antigens, thus negating the need for adjuvants.
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Glicoproteínas Hemaglutininas del Virus de la Influenza , Vacunas contra la Influenza , Animales , Anticuerpos Antivirales , Antígenos Bacterianos , Antígenos Virales/genética , Vacunas Bacterianas , Células CHO , Cricetinae , Cricetulus , Lampreas/genética , Ratones , Ratones Endogámicos BALB CRESUMEN
After decades of inconsequential infections, and sporadic outbreaks in the Asia-Pacific region between 2007 and 2013, Zika virus caused a widespread epidemic in South America in 2015 that was complicated by severe congenital infections. After the WHO declared a Public Health Emergency of International Concern in February 2016, vaccine development efforts based on different platforms were initiated. Several candidates have since been evaluated in clinical phase I studies. Of these, a Zika purified inactivated vaccine (ZPIV), adjuvanted with aluminum hydroxide, developed by the Walter Reed Army Institute of Research (WRAIR), yielded high seroconversion rates. Sanofi Pasteur further optimized the vaccine in terms of production scale, purification conditions and regulatory compliance, using its experience in flavivirus vaccine development. Here we report that the resulting optimized vaccine (ZPIV-SP) elicited robust seroneutralizing antibody responses and provided complete protection from homologous Zika virus strain challenge in immunocompetent BALB/c mice. ZPIV-SP also showed improved immunogenicity compared with the first-generation vaccine, and improved efficacy in the more permissive interferon receptor-deficient A129 mice. Finally, analysis of the IgG response directed towards nonstructural protein 1 (NS1) suggests that viral NS1 was efficiently removed during the optimized purification process of ZPIV-SP. Together, these results suggest that the optimized vaccine is well suited for further evaluation in larger animal models and late-stage clinical studies.
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Inmunogenicidad Vacunal , Potencia de la Vacuna , Vacunas de Productos Inactivados/inmunología , Vacunas Virales/inmunología , Infección por el Virus Zika/inmunología , Virus Zika/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Ratones , Ratones Noqueados , Proteínas del Envoltorio Viral/inmunología , Virus Zika/genética , Infección por el Virus Zika/prevención & control , Infección por el Virus Zika/virologíaRESUMEN
Zika virus (ZIKV) is an emerging mosquito-borne pathogen representing a global health concern. It has been linked to fetal microcephaly and other birth defects and neurological disorders in adults. Sanofi Pasteur has engaged in the development of an inactivated ZIKV vaccine, as well as a live chimeric vaccine candidate ChimeriVax-Zika (CYZ) that could become a preferred vaccine depending on future ZIKV epidemiology. This report focuses on the CYZ candidate that was constructed by replacing the pre-membrane and envelope (prM-E) genes in the genome of live attenuated yellow fever 17D vaccine virus (YF 17D) with those from ZIKV yielding a viable CYZ chimeric virus. The replication rate of CYZ in the Vero cell substrate was increased by using a hybrid YF 17D-ZIKV signal sequence for the prM protein. CYZ was highly attenuated both in mice and in human in vitro models (human neuroblastoma and neuronal progenitor cells), without the need for additional attenuating modifications. It exhibited significantly reduced viral loads in organs compared to a wild-type ZIKV and a complete lack of neuroinvasion following inoculation of immunodeficient A129 mice. A single dose of CYZ elicited high titers of ZIKV-specific neutralizing antibodies in both immunocompetent and A129 mice and protected animals from ZIKV challenge. The data indicate that CYZ is a promising vaccine candidate against ZIKV.
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Vacunas Atenuadas/inmunología , Vacunas Virales/inmunología , Virus de la Fiebre Amarilla/inmunología , Infección por el Virus Zika/prevención & control , Virus Zika/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Línea Celular , Chlorocebus aethiops , Humanos , Ratones , Ratones Endogámicos ICR , Vacunas Atenuadas/uso terapéutico , Células Vero , Carga Viral , Vacunas Virales/uso terapéutico , Infección por el Virus Zika/inmunologíaRESUMEN
HSV-2 vaccine is needed to prevent genital disease, latent infection, and virus transmission. A replication-deficient mutant virus (dl5-29) has demonstrated promising efficacy in animal models of genital herpes. However, the immunogenicity, protective efficacy, and non-replicative status of the highly purified clinical vaccine candidate (HSV529) derived from dl5-29 have not been evaluated. Humoral and cellular immune responses were measured in mice and guinea pigs immunized with HSV529. Protection against acute and recurrent genital herpes, mortality, latent infection, and viral shedding after vaginal HSV-2 infection was determined in mice or in naïve and HSV-1 seropositive guinea pigs. HSV529 replication and pathogenicity were investigated in three sensitive models of virus replication: severe combined immunodeficient (SCID/Beige) mice inoculated by the intramuscular route, suckling mice inoculated by the intracranial route, and vaginally-inoculated guinea pigs. HSV529 immunization induced HSV-2-neutralizing antibody production in mice and guinea pigs. In mice, it induced production of specific HSV-2 antibodies and splenocytes secreting IFNγ or IL-5. Immunization effectively prevented HSV-2 infection in all three animal models by reducing mortality, acute genital disease severity and frequency, and viral shedding. It also reduced ganglionic viral latency and recurrent disease in naïve and HSV-1 seropositive guinea pigs. HSV529 replication/propagation was not detected in the muscles of SCID/Beige mice, in the brains of suckling mice, or in vaginal secretions of inoculated guinea pigs. These results confirm the non-replicative status, as well as its immunogenicity and efficacy in mice and guinea pigs, including HSV-1 seropositive guinea pigs. In mice, HSV529 produced Th1/Th2 characteristic immune response thought to be necessary for an effective vaccine. These results further support the clinical investigation of HSV529 in human subjects as a prophylactic vaccine.
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Anticuerpos Neutralizantes/biosíntesis , Anticuerpos Antivirales/biosíntesis , Herpes Genital/prevención & control , Vacunas contra el Virus del Herpes Simple/administración & dosificación , Herpesvirus Humano 2/inmunología , Proteínas del Envoltorio Viral/inmunología , Animales , Femenino , Cobayas , Herpes Genital/inmunología , Herpes Genital/mortalidad , Herpes Genital/virología , Vacunas contra el Virus del Herpes Simple/genética , Vacunas contra el Virus del Herpes Simple/inmunología , Herpesvirus Humano 2/genética , Humanos , Inmunidad Celular/efectos de los fármacos , Inmunidad Humoral/efectos de los fármacos , Inmunización , Interferón gamma/biosíntesis , Interferón gamma/metabolismo , Interleucina-5/biosíntesis , Interleucina-5/metabolismo , Ratones , Ratones SCID , Análisis de Supervivencia , Balance Th1 - Th2/efectos de los fármacos , Vagina/efectos de los fármacos , Vagina/inmunología , Vagina/virología , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Latencia del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Esparcimiento de Virus/efectos de los fármacosRESUMEN
The membrane displayed antigen haemagglutinin (HA) from several influenza strains were expressed in the Leishmania tarentolae system. This non-conventional expression system based on a parasite of lizards, can be readily propagated to high cell density (>10(8)cells/mL) in a simple incubator at 26°C. The genes encoding HA proteins were cloned from six influenza strains, among these being a 2009 A/H1N1 pandemic strain from swine origin, namely A/California/07/09(H1N1). Soluble HA proteins were secreted into the cell culture medium and were easily and successfully purified via a His-Tag domain fused to the proteins. The overall process could be conducted in less than 3 months and resulted in a yield of approximately 1.5-5mg of HA per liter of biofermenter culture after purification. The recombinant HA proteins expressed by L. tarentolae were characterized by dynamic light scattering and were observed to be mostly monomeric. The L. tarentolae recombinant HA proteins were immunogenic in mice at a dose of 10µg when administered twice with an oil-in-water emulsion-based adjuvant. These results suggest that the L. tarentolae expression system may be an alternative to the current egg-based vaccine production.
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Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Vacunas contra la Influenza/inmunología , Leishmania/metabolismo , Infecciones por Orthomyxoviridae/prevención & control , Animales , Anticuerpos Antivirales/sangre , Clonación Molecular , Femenino , Pruebas de Inhibición de Hemaglutinación , Glicoproteínas Hemaglutininas del Virus de la Influenza/biosíntesis , Inmunoglobulina G/sangre , Subtipo H1N1 del Virus de la Influenza A , Ratones Endogámicos BALB C , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunologíaRESUMEN
Two IgM monoclonal antibodies (MAbs), Y6F5 and Y13F9, were selected during a screening of clones obtained immunising BALB/c mice with purified envelop proteins of the A/Sydney/5/97 (H3N2) IVR108 influenza strain. These MAbs recognised avian glycans on the haemagglutinin (HA) of the virus. This broad recognition allowed these MAbs to be used as enzyme-labelled secondary antibody reagents in a strain specific enzyme-linked immunosorbent assay (ELISA) in combination with a capture MAb that recognised and allowed the quantitation of the strain specific HA protein present in an egg-produced influenza vaccine. Advantage was taken of these MAbs to develop a universal ELISA in which the MAbs were used both as capture antibody and as enzyme-labelled secondary antibody to detect and quantify the HA protein of any egg-derived influenza vaccine. These avian-glycan specific IgM MAbs may prove to be particularly useful for determining the HA concentration in monovalent egg-derived pandemic influenza vaccines, in which the HA concentration may be lower than 5µg/ml. The HA detection limit in the ELISA assays developed in this study was 1.9µg/ml, as opposed to the 5µg/ml quantitation limit generally accepted for the standard single-radial-immunodiffusion (SRID) assay, the approved technique for quantifying HA content in influenza vaccines. These ELISAs can also be used to quantify influenza HA formulated with emulsion-based or mineral salt adjuvants that could interfere with HA measurement by the SRID assay.
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Anticuerpos Monoclonales , Antígenos Virales/análisis , Glicoproteínas Hemaglutininas del Virus de la Influenza/análisis , Inmunoglobulina M , Virus de la Influenza A/crecimiento & desarrollo , Vacunas contra la Influenza/química , Polisacáridos/inmunología , Animales , Antígenos Virales/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Inmunodifusión/métodos , Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Ratones , Ratones Endogámicos BALB CRESUMEN
Pandemic influenza vaccines have been manufactured using the A/California/07/2009 (H1N1) strain as recommended by the World Health Organization. We evaluated in mice the immunogenicity of pandemic (H1N1) 2009 vaccine and the impact of prior vaccination against seasonal trivalent influenza vaccines (TIV) on antibody responses against pandemic (H1N1) 2009. In naïve mice, a single dose of unadjuvanted H1N1 vaccine (3 microg of HA) was shown to elicit hemagglutination inhibition (HI) antibody titers >40, a titer associated with protection in humans against seasonal influenza. A second vaccine dose of pandemic (H1N1) 2009 vaccine strongly increased these titers, which were consistently higher in mice previously primed with TIV than in naïve mice. At a low immunization dose (0.3 microg of HA), the AF03-adjuvanted vaccine elicited higher HI antibody titers than the corresponding unadjuvanted vaccines in both naïve and TIV-primed animals, suggesting a potential for antigen dose-sparing. These results are in accordance with the use in humans of a split-virion inactivated pandemic (H1N1) 2009 vaccine formulated with or without AF03 adjuvant to protect children and young adults against influenza A (H1N1) 2009 infection.