Most of the escaped farmed salmon entering a river during a 5-year period were infected with one or more viruses.

Disease interactions between farmed and wild populations have been poorly documented for most aquaculture species, in part due to the complexities to study this. Here, we tested 567 farmed Atlantic salmon escapees, captured in a Norwegian river during 2014-2018, for five viral infections that are prevalent in global salmonid aquaculture. Over 90% of the escapees were infected with one or more viruses. Overall prevalences were: 75.7% for piscine orthoreovirus (PRV-1), 43.6% for salmonid alphavirus (SAV), 31.2% for piscine myocarditis virus (PMCV), 1.2% for infectious pancreatic necrosis virus (IPNV) and 0.4% for salmon anaemia virus (ISAV). A significantly higher prevalence of PMCV infection was observed in immature compared to mature individuals. The prevalence of both SAV and PMCV infections was higher in fish determined by fatty acid profiling to be 'recent' as opposed to 'early' escapees that had been in the wild for a longer period of time. This is the first study to establish a time-series of viral infection status of escapees entering a river with a native salmon population. Our results demonstrate that farmed escapees represent a continuous source of infectious agents which could potentially be transmitted to wild fish populations.

Occurrence of salmonid alphavirus and piscine orthoreovirus-1 infections in migrating salmon (Salmo salar L.) post-smolt in western Norway.

Viral diseases are a serious problem in Atlantic salmon (Salmo salar L.) farming in Norway, often leading to reduced fish welfare and increased mortality. Disease outbreaks in salmon farms may lead to spread of viruses to the surrounding environment. There is a public concern that viral diseases may negatively affect the wild salmon populations. Pancreas disease (PD) caused by salmonid alphavirus (SAV) and heart and skeletal muscle inflammation (HSMI) caused by piscine orthoreovirus-1 (PRV-1) are common viral diseases in salmon farms in western Norway. In the current study, we investigated the occurrence of SAV and PRV-1 infections in 651 migrating salmon post-smolt collected from three fjord systems (Sognefjorden, Osterfjorden and Hardangerfjorden) located in western Norway in 2013 and 2014 by real-time RT-PCR. Of the collected post-smolts, 303 were of wild origin and 348 were hatchery-released. SAV was not detected in any of the tested post-smolt, but PRV-1 was detected in 4.6% of them. The Ct values of PRV-1 positive fish were usually high (mean 32.0; range: 20.1-36.8). PRV-1 prevalence in post-smolts from the three fjords was 6.1% in Sognefjorden followed by 4.8% in Osterfjorden and 2.3% in Hardangerfjorden. The prevalence PRV-1 was significantly higher in wild (6.9%) compared to hatchery-released post-smolt (2.6%). The occurrence of PRV-1 infection in the fish was lowest in the Hardangerfjorden which has the highest fish farming intensity. Our results suggest that SAV infection are uncommon in migrating smolt while PRV-1 infection can be detected at low level. These findings suggest that migrating smolts were at low risk from SAV or PRV-1 released from salmon farms located in their migration routes in 2013 and 2014.

Co-diversification of an intestinal Mycoplasma and its salmonid host.

Understanding the evolutionary relationships between a host and its intestinal resident bacteria can transform how we understand adaptive phenotypic traits. The interplay between hosts and their resident bacteria inevitably affects the intestinal environment and, thereby, the living conditions of both the host and the microbiota. Thereby this co-existence likely influences the fitness of both bacteria and host. Whether this co-existence leads to evolutionary co-diversification in animals is largely unexplored, mainly due to the complexity of the environment and microbial communities and the often low host selection. We present the gut metagenome from wild Atlantic salmon (Salmo salar), a new wild organism model with an intestinal microbiota of low complexity and a well-described population structure, making it well-suited for investigating co-evolution. Our data reveal a strong host selection of a core gut microbiota dominated by a single Mycoplasma species. We found a clear co-diversification between the population structure of Atlantic salmon and nucleotide variability of the intestinal Mycoplasma populations conforming to expectations from co-evolution between host and resident bacteria. Our results show that the stable microbiota of Atlantic salmon has evolved with its salmonid host populations while potentially providing adaptive traits to the salmon host populations, including defence mechanisms, biosynthesis of essential amino acids, and metabolism of B vitamins. We highlight Atlantic salmon as a novel model for studying co-evolution between vertebrate hosts and their resident bacteria.

Negative impacts of the sea lice prophylactic emamectin benzoate on the survival of hatchery released salmon smolts in rivers.

Emamectin benzoate (EB) is a prophylactic pharmaceutical used to protect Atlantic salmon (Salmo salar) smolts migrating out of rivers and into the ocean against sea lice parasites. Randomized control trials comparing the marine survival of smolts treated with EB to a control group is used to calculate the fraction of marine mortality attributable to sea lice parasitism. However, it is assumed that there is no baseline difference in survival induced by the application of EB treatment. We used a combined laboratory and field study approach to investigate the potential impacts of EB treatment on behaviour and survival of hatchery-reared Atlantic salmon in western Norway. In aquaria experiments, EB-treated salmon smolts did not differ significantly in exploratory behaviour. Fish from treated groups responded similarly to simulated predator attack with spontaneous escape and elevated gill beat rate. Three rivers in the Osterfjord system of western Norway were selected for field experiments, Dale, Vosso, and Modalen. Dale River smolts were treated with intraperitoneal EB injections and had lower probability of detection in a wolf trap downstream of the release site than control smolts. Salmon smolts raised in the Vosso River hatchery were treated with EB delivered in their food and were detected on PIT antennas at the rivermouth of Vosso and Modalen at lower rates than control fish, but only when released at downstream sites. Calculation of risk ratios suggested that the bias in mortality caused by treatment with EB decreased the estimated survival of treated fish from an expected 18%to 46%, reducing the observable negative impact of sea lice on Atlantic salmon smolts in randomized control trials. The results suggest that estimates of the fraction of mortality attributable to sea lice may be underestimated due to lower baseline survival of treated fish caused by treatment and bring urgent attention towards a potential systematic underestimation of the impacts of sea lice on wild salmon.

Lower levels of Persistent Organic Pollutants, metals and the marine omega 3-fatty acid DHA in farmed compared to wild Atlantic salmon (Salmo salar).

Contaminants and fatty acid levels in farmed- versus wild Atlantic salmon have been a hot topic of debate in terms of food safety. The present study determined dioxins (polychlorinated dibenzo-p-dioxin and dibenzofuran), polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), organochlorine pesticides (OCPs), metals and fatty acids in wild and farmed Atlantic salmon. Contaminant levels of dioxins, PCBs, OCPs (DDT, dieldrin, lindane, chlordane, Mirex, and toxaphene), and mercury were higher in wild salmon than in farmed salmon, as were the concentrations of the essential elements selenium, copper, zinc and iron, and the marine omega-3 fatty acid docosahexaenoic acid (DHA). PBDE, endosulfan, pentachlorobenzene, hexachlorobenzene, cadmium and lead levels were low and comparable in both wild and farmed fish, and there was no significant difference in the marine omega-3 fatty acid eicosapentaenoic acid (EPA) concentration. The total fat content was significantly higher in farmed than wild salmon due to a higher content of both saturated and monounsaturated fatty acids, as well as a higher content of omega-6 fatty acids. The omega-3 to omega-6 fatty acid ratio was considerably lower in farmed than wild salmon due to the high level of omega-6 fatty acids. Contaminant concentrations in Atlantic salmon were well below maximum levels applicable in the European Union. Atlantic salmon, both farmed and wild, is a good source of EPA and DHA with a 200g portion per week contributing 3.2g or 2.8g respectively, being almost twice the intake considered adequate for adults by the European Food Safety Authority (i.e. 250mg/day or 1.75g/week).

Occurrence of salmonid alphavirus (SAV) and piscine orthoreovirus (PRV) infections in wild sea trout Salmo trutta in Norway.

Viral diseases represent a serious problem in Atlantic salmon (Salmo salar L.) farming in Norway. Pancreas disease (PD) caused by salmonid alphavirus (SAV) and heart and skeletal muscle inflammation (HSMI) caused by piscine orthoreovirus (PRV) are among the most frequently diagnosed viral diseases in recent years. The possible spread of viruses from salmon farms to wild fish is a major public concern. Sea trout S. trutta collected from the major farming areas along the Norwegian coast are likely to have been exposed to SAV and PRV from farms with disease outbreaks. We examined 843 sea trout from 4 counties in Norway for SAV and PRV infections. We did not detect SAV in any of the tested fish, although significant numbers of the trout were caught in areas with frequent PD outbreaks. Low levels of PRV were detected in 1.3% of the sea trout. PRV-infected sea trout were caught in both salmon farming and non-farming areas, so the occurrence of infections was not associated with farming intensity or HSMI cases. Our results suggest that SAV and PRV infections are uncommon in wild sea trout. Hence, we found no evidence that sea trout are at risk from SAV or PRV released from salmon farms.

The frequency of spontaneous triploidy in farmed Atlantic salmon produced in Norway during the period 2007-2014.

BACKGROUND: Spontaneous triploidy has been reported in a number of fish species, and is often linked with in vivo or in vitro ageing of eggs post ovulation. Here, we provide the first investigation into the frequency of spontaneous triploidy in farmed Atlantic salmon by analysing more than 4000 fish from 55 farms, and approximately 1000 recaptured escapees, all sampled in the period 2007-2014. In addition, we compare microsatellite genotyping against flow cytometry and red blood cell diameter in a set of 45 putatively diploid and 45 putatively triploid Atlantic salmon. RESULTS: The three methods implemented for ploidy determination gave consistent results, thus validating the methods used here. Overall, 2.0% spontaneous triploids were observed in salmon sampled on farms. The frequency of spontaneous triploids varied greatly among sea cages (0-28%), but they were observed in similar frequencies among the three primary breeding companies (1.8-2.4%). Spontaneous triploids were observed in all farming regions in Norway, and in all years sampled. Spontaneous triploids were also observed among the escapees recaptured in both the marine environment and in rivers. CONCLUSIONS: Spontaneous triploidy in commercially produced Atlantic salmon is likely to be a result of the practices employed by the industry. For logistical reasons, there is sometimes a pause of hours, and in some cases overnight, between killing the female broodfish, removal of her eggs, and fertilization. This gives the eggs time to age post ovulation, and increases the probability of duplication of the maternal chromosome set by inhibition of the second polar body release after normal meiosis II in the oocyte.

T-helper 1 cells elicited by H5N1 vaccination predict seroprotection.

BACKGROUND: Vaccination is the best measure to protect the population against a potential influenza H5N1 pandemic, but 2 doses of vaccine are needed to elicit protective immune responses. An immunological marker for H5N1 vaccine effectiveness is needed for early identification of the best vaccine candidate. METHODS: We conducted a phase I clinical trial of a virosomal H5N1 vaccine adjuvanted with Matrix M. Sixty adult volunteers were vaccinated intramuscularly with 2 doses of either 30 µg hemagglutinin (HA) alone or with 1.5, 7.5, or 30 µg HA and Matrix M adjuvant (50 µg). The humoral response was measured by the hemagglutination inhibition (HI), microneutralization (MN), and single radial hemolysis (SRH) assays, and the CD4(+) T-helper 1 (Th1)-cell response was measured by intracellular staining for the cytokines interleukin 2, interferon γ, and tumor necrosis factor α. RESULTS: The adjuvanted vaccine effectively induced CD4(+) Th1-cell responses, and the frequency of influenza-specific Th1 cells after the first vaccine dose predicted subsequent HI, MN, and SRH seroprotective responses after the second vaccination. CONCLUSIONS: These results support early identification of Th1-cell responses as a predictive biomarker for an efficient vaccine response, which could have great implications for early identification of persons with low or no response to vaccine when evaluating future pandemic influenza vaccines.

The human potential of a recombinant pandemic influenza vaccine produced in tobacco plants.

Rapid production of influenza vaccine antigen is an important challenge when a new pandemic occurs. Production of recombinant antigens in plants is a quick, cost effective and up scalable new strategy for influenza vaccine production. In this study, we have characterized a recombinant influenza haemagglutinin antigen (HAC1) that was derived from the 2009 pandemic H1N1 (pdmH1N1) virus and expressed in tobacco plants. Volunteers vaccinated with the 2009 pdmH1N1 oil-in-water adjuvanted vaccine provided serum and lymphocyte samples that were used to study the immunogenic properties of the HAC1 antigen in vitro. By 7 d post vaccination, the vaccine fulfilled the licensing criteria for antibody responses to the HA detected by haemagglutination inhibition and single radial hemolysis. By ELISA and ELISPOT analysis we showed that HAC1 was recognized by specific serum antibodies and antibody secreting cells, respectively. We conducted a kinetic analysis and found a peak of serum HAC1 specific antibody response between day 14 and 21 post vaccination by ELISA. We also detected elevated production of IL-2 and IFNγ and low frequencies of CD4(+) T cells producing single or multiple Th1 cytokines after stimulating PBMCs (peripheral blood mononuclear cells) with the HAC1 antigen in vitro. This indicates that the antigen can interact with T cells, although confirming an effective adjuvant would be required to improve the T-cell stimulation of plant based vaccines. We conclude that the tobacco derived recombinant HAC1 antigen is a promising vaccine candidate recognized by both B- and T cells.

The mucosal and systemic immune responses elicited by a chitosan-adjuvanted intranasal influenza H5N1 vaccine.

BACKGROUND: Development of influenza vaccines that induce mucosal immunity has been highlighted by the World Health Organisation as a priority (Vaccine 2005;23:1529). Dose-sparing strategies and an efficient mass-vaccination regime will be paramount to reduce the morbidity and mortality of a future H5N1 pandemic. OBJECTIVES: This study has investigated the immune response and the dose-sparing potential of a chitosan-adjuvanted intranasal H5N1 (RG-14) subunit (SU) vaccine in a mouse model. METHODS: Groups of mice were intranasally immunised once or twice with a chitosan (5 mg/ml)-adjuvanted SU vaccine [7·5, 15 or 30 µg haemagglutinin (HA)] or with a non-adjuvanted SU vaccine (30 µg HA). For comparison, another group of mice were intranasally immunised with a whole H5N1 (RG-14) virus (WV) vaccine (15 µg HA), and the control group consisted of unimmunised mice. RESULTS: The chitosan-adjuvanted SU vaccine induced an immune response superior to that of the non-adjuvanted SU vaccine. Compared with the non-adjuvanted SU group, the chitosan-adjuvanted SU vaccine elicited higher numbers of influenza-specific antibody-secreting cells (ASCs), higher concentrations of local and systemic antibodies and correspondingly an improved haemagglutination inhibition (HI) and single radial haemolysis (SRH) response against both the homologous vaccine strain and drifted H5 strains. We measured a mixed T-helper 1/T-helper 2 cytokine response in the chitosan-adjuvanted SU groups, and these groups had an increased percentage of virus-specific CD4(+) T cells producing two Thelper 1 (Th1) cytokines simultaneously compared with the non-adjuvanted SU group. Overall, the WV vaccine induced higher antibody concentrations in sera and an HI and SRH response similar to that of the chitosan-adjuvanted SU vaccine. Furthermore, the WV vaccine formulation showed a stronger bias towards a T-helper 1 profile than the SU vaccine and elicited the highest frequencies of CD4(+) Th1 cells simultaneously secreting three different cytokines (INFγ(+) , IL2(+) and INFα(+) ). As expected, two immunisations gave a better immune response than one in all groups. The control group had very low or not detectable results in the performed immunoassays. CONCLUSION: The cross-clade serum reactivity, improved B- and T-cell responses and dose-sparing potential of chitosan show that a chitosan-adjuvanted intranasal influenza vaccine is a promising candidate vaccine for further preclinical development.

Evaluation of a virosomal H5N1 vaccine formulated with Matrix M™ adjuvant in a phase I clinical trial.

The avian influenza H5 virus epizootic continues to cause zoonosis with human fatalities, highlighting the continued need for pandemic preparedness against this subtype. This study evaluated the tolerability and immunogenicity of a Matrix M™ adjuvanted virosomal H5N1 vaccine in a phase I clinical trial. Sixty healthy adults were vaccinated intramuscularly with two doses of influenza H5N1 (NIBRG-14) virosomal vaccine alone (30 µg haemagglutinin (HA)) or 1.5, 7.5 or 30 µg HA formulated with 50 µg Matrix M™ adjuvant. The antibody response was analysed by haemagglutination inhibition (HI), microneutralisation (MN) and single radial haemolysis (SRH) assays. The vaccine was well tolerated in all groups but injection site pain was more frequently observed in the Matrix M™ adjuvanted groups. The vaccine elicited homologous and heterologous H5N1-specific antibody responses and the Matrix M™ adjuvanted formulations met all the EU regulatory criteria. In conclusion, Matrix M™ adjuvant was well tolerated and augmented the antibody response allowing considerable dose sparing down to 1.5 µg HA.

Matrix-M adjuvanted virosomal H5N1 vaccine confers protection against lethal viral challenge in a murine model.

BACKGROUND: A candidate pandemic influenza H5N1 vaccine should provide rapid and long-lasting immunity against antigenically drifted viruses. As H5N1 viruses are poorly immunogenic, this may require a combination of immune potentiating strategies. An attractive approach is combining the intrinsic immunogenicity of virosomes with another promising adjuvant to further boost the immune response. As regulatory authorities have not yet approved a surrogate correlate of protection for H5N1 vaccines, it is important to test the protective efficacy of candidate H5N1 vaccines in a viral challenge study. OBJECTIVES: This study investigated in a murine model the protective efficacy of Matrix-M adjuvanted virosomal influenza H5N1 vaccine against highly pathogenic lethal viral challenge. METHODS: Mice were vaccinated intranasally (IN) or intramuscularly (IM) with 7·5 µg and 30 µg HA of inactivated A/Vietnam/1194/2004 (H5N1) (NIBRG-14) virosomal adjuvanted vaccine formulated with or without 10 µg of Matrix-M adjuvant and challenged IN with the highly pathogenic A/Vietnam/1194/2004 (H5N1) virus. RESULTS AND CONCLUSIONS: IM vaccination provided protection irrespective of dose and the presence of Matrix-M adjuvant, whilst the IN vaccine required adjuvant to protect against the challenge. The Matrix-M adjuvanted vaccine induced a strong and cross-reactive serum antibody response indicative of seroprotection after both IM and IN administration. In addition, the IM vaccine induced the highest frequencies of influenza specific CD4+ and CD8+ T-cells. The results confirm a high potential of Matrix-M adjuvanted virosomal vaccines and support the progress of this vaccine into a phase 1 clinical trial.

Intranasal c-di-GMP-adjuvanted plant-derived H5 influenza vaccine induces multifunctional Th1 CD4+ cells and strong mucosal and systemic antibody responses in mice.

Vaccination is the best available measure of limiting the impact of the next influenza pandemic. Ideally, a candidate pandemic influenza vaccine should be easy to administer and should elicit strong mucosal and systemic immune responses. Production of influenza subunit antigen in transient plant expression systems is an alternative to overcome the bottleneck in vaccine supply during influenza pandemic. Furthermore, a needle-free intranasal influenza vaccine is an attractive approach, which may provide immunity at the portal of virus entry. The present study investigated the detailed humoral and cellular immune responses in mice vaccinated intranasally or intramuscularly with plant-derived influenza H5N1 (A/Anhui/1/05) antigen alone or formulated with bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) as adjuvant. The use of c-di-GMP as intramuscular adjuvant did not enhance the immune response to plant-derived influenza H5 antigen. However, intranasal c-di-GMP-adjuvanted vaccine induced strong mucosal and systemic humoral immune responses. Additionally, the intranasal vaccine elicited a balanced Th1/Th2 profile and, most importantly, high frequencies of multifunctional Th1 CD4(+) cells. Our results highlight that c-di-GMP is a promising mucosal adjuvant for pandemic influenza vaccine development.

An adjuvanted pandemic influenza H1N1 vaccine provides early and long term protection in health care workers.

Mass vaccination was the most effective prophylaxis for protecting the population during the influenza H1N1 pandemic. We have evaluated the tolerability, immunogenicity and kinetics of the antibody response to a monovalent oil-in-water (AS03) adjuvanted human pandemic split influenza A/California/7/2009 H1N1 (3.75 µg haemagglutinin) vaccine in health care workers. Vaccination elicited a rapid and early protective level of haemagglutination inhibition antibody from 6 to 7 days post vaccination, and by 14 to 21 days post vaccination, up to 98% of vaccinees had protective antibody titres which persisted for at least 3 months in 84-92% of subjects. A rapid induction of protective antibody is important in reducing community spread of pandemic influenza and in helping maintain the integrity of the health care system during the pandemic.

Intramuscular Matrix-M-adjuvanted virosomal H5N1 vaccine induces high frequencies of multifunctional Th1 CD4+ cells and strong antibody responses in mice.

Ideally, a candidate pandemic influenza vaccine should elicit rapid and strong cell-mediated and humoral immune responses, which are long-lasting and exhibit broad cross-reactivity against drifted strains. The present study investigated the detailed humoral and cellular immune responses in mice vaccinated intranasally or intramuscularly with inactivated influenza H5N1 (NIBRG-14) virosomal vaccine alone or formulated with Matrix-M adjuvant. The intramuscular Matrix-M-adjuvanted vaccine induced a strong immediate and long-term humoral immune response with high cross-reactivity against drifted H5N1 viruses and showed a dose-sparing potential. Additionally, the vaccine induced a balanced Th1/Th2 cytokine profile and most importantly high frequencies of multifunctional Th1 CD4(+) cells. Our results highlight that Matrix-M adjuvant is a promising parenteral adjuvant for formulating pandemic candidate vaccines.

A cell-based H7N1 split influenza virion vaccine confers protection in mouse and ferret challenge models.

BACKGROUND: In recent years, several avian influenza subtypes (H5, H7 and H9) have transmitted directly from birds to man, posing a pandemic threat. OBJECTIVES: We have investigated the immunogenicity and protective efficacy of a cell based candidate pandemic influenza H7 vaccine in pre-clinical animal models. METHODS: Mice and ferrets were immunised with two doses of the split virus vaccine (12-24 microg haemagglutinin) with or without aluminium hydroxide adjuvant and challenged 3 weeks after second dose with the highly pathogenic A/chicken/Italy/13474/99 (H7N1) virus. The H7N1-specific serum antibody response was also measured. After challenge, viral shedding, weight loss, disease signs and death (only mice) were recorded. RESULTS: Low-to-modest serum antibody titres were detected after vaccination. Nevertheless, the vaccine induced significant protection from disease after challenge with the wild-type virus. In the murine lethal challenge model, vaccination effectively prevented death and, furthermore, formulation with adjuvant reduced excessive weight loss and viral shedding. In ferrets, vaccination reduced viral shedding and protected against systemic spread of the virus. CONCLUSIONS: We have extended to the H7 subtype the finding that protective efficacy may not be directly correlated with the pre-challenge levels of serum antibodies, a finding which could be of great importance in assessing the potential effectiveness of pandemic influenza vaccines.

A phase I clinical trial of a PER.C6 cell grown influenza H7 virus vaccine.

Avian influenza H7 viruses have transmitted from poultry to man causing human illness and fatality, highlighting the need for pandemic preparedness against this subtype. We have developed and tested the first cell-based human vaccine against H7 avian influenza virus in a phase I clinical trial. Sixty healthy volunteers were intramuscularly vaccinated with two doses of split H7N1 virus vaccine containing 12 microg or 24 microg haemagglutinin alone or with aluminium hydroxide adjuvant (300 microg or 600 microg, respectively). The vaccine was well tolerated in all subjects and no serious adverse events occurred. The vaccine elicited low haemagglutination inhibition and microneutralisation titres, although the addition of aluminium adjuvant augmented the antibody response. We found a higher number of antibody secreting cells and an association with IL-2 production in subjects with antibody response. In conclusion, our study shows that producing effective H7 pandemic vaccines is as challenging as has been observed for H5 vaccines.

Pandemic influenza vaccines - the challenges.

Recent years' enzootic spread of highly pathogenic H5N1 virus among poultry and the many lethal zoonoses in its wake has stimulated basic and applied pandemic vaccine research. The quest for an efficacious, affordable and timely accessible pandemic vaccine has been high on the agenda. When a variant H1N1 strain of swine origin emerged as a pandemic virus, it surprised many, as this subtype is well-known to man as a seasonal virus. This review will cover some difficult vaccine questions, such as the immunological challenges, the new production platforms, and the limited supply and global equity issues.

Quality and kinetics of the antibody response in mice after three different low-dose influenza virus vaccination strategies.

The threat of a new influenza pandemic has led to renewed interest in dose-sparing vaccination strategies such as intradermal immunization and the use of adjuvanted vaccines. In this study we compared the quality and kinetics of the serum antibody response elicited in mice after one or two immunizations with a split influenza A (H3N2) virus, using three different low-dose vaccination strategies. The mice were divided into four groups, receiving either a low-dose vaccine (3 microg hemagglutinin [HA]) intradermally or intramuscularly with or without aluminum adjuvant or the normal human vaccine dose (15 microg HA) intramuscularly. Sera were collected weekly after vaccination and tested in the hemagglutination inhibition, virus neutralization, and enzyme-linked immunosorbent assays. The antibody responses induced after intradermal or intramuscular low-dose vaccinations were similar and lower than those observed after the human vaccine dose. However, low-dose adjuvanted vaccine elicited a serum antibody response comparable to that elicited by the human dose, although the second immunization did not result in any increase in cross-reactive hemagglutination inhibition antibodies, and the peak serum antibody response was observed 1 week later than in the other vaccination groups. Our murine data suggest that the low-dose intradermal route does not show any obvious advantage over the low-dose intramuscular route in inducing a serum antibody response and that none of the low-dose vaccination strategies is as effective as intramuscular vaccination with the normal human dose. However, the low-dose aluminum-adjuvanted vaccine could present a feasible alternative in case of limited vaccine supply.