A tetravalent virus-like particle vaccine designed to display domain III of dengue envelope proteins induces multi-serotype neutralizing antibodies in mice and macaques which confer protection against antibody dependent enhancement in AG129 mice.

BACKGROUND: Dengue is one of the fastest spreading vector-borne diseases, caused by four antigenically distinct dengue viruses (DENVs). Antibodies against DENVs are responsible for both protection as well as pathogenesis. A vaccine that is safe for and efficacious in all people irrespective of their age and domicile is still an unmet need. It is becoming increasingly apparent that vaccine design must eliminate epitopes implicated in the induction of infection-enhancing antibodies. METHODOLOGY/PRINCIPAL FINDINGS: We report a Pichia pastoris-expressed dengue immunogen, DSV4, based on DENV envelope protein domain III (EDIII), which contains well-characterized serotype-specific and cross-reactive epitopes. In natural infection, <10% of the total neutralizing antibody response is EDIII-directed. Yet, this is a functionally relevant domain which interacts with the host cell surface receptor. DSV4 was designed by in-frame fusion of EDIII of all four DENV serotypes and hepatitis B surface (S) antigen and co-expressed with unfused S antigen to form mosaic virus-like particles (VLPs). These VLPs displayed EDIIIs of all four DENV serotypes based on probing with a battery of serotype-specific anti-EDIII monoclonal antibodies. The DSV4 VLPs were highly immunogenic, inducing potent and durable neutralizing antibodies against all four DENV serotypes encompassing multiple genotypes, in mice and macaques. DSV4-induced murine antibodies suppressed viremia in AG129 mice and conferred protection against lethal DENV-4 virus challenge. Further, neither murine nor macaque anti-DSV4 antibodies promoted mortality or inflammatory cytokine production when passively transferred and tested in an in vivo dengue disease enhancement model of AG129 mice. CONCLUSIONS/SIGNIFICANCE: Directing the immune response to a non-immunodominant but functionally relevant serotype-specific dengue epitope of the four DENV serotypes, displayed on a VLP platform, can help minimize the risk of inducing disease-enhancing antibodies while eliciting effective tetravalent seroconversion. DSV4 has a significant potential to emerge as a safe, efficacious and inexpensive subunit dengue vaccine candidate.

Immunogenicity and efficacy of alphavirus-derived replicon vaccines for respiratory syncytial virus and human metapneumovirus in nonhuman primates.

Human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV) are major causes of illness among children, the elderly, and the immunocompromised. No vaccine has been licensed for protection against either of these viruses. We tested the ability of two Venezuelan equine encephalitis virus-based viral replicon particle (VEE-VRP) vaccines that express the hRSV or hMPV fusion (F) protein to confer protection against hRSV or hMPV in African green monkeys. Animals immunized with VEE-VRP vaccines developed RSV or MPV F-specific antibodies and serum neutralizing activity. Compared to control animals, immunized animals were better able to control viral load in the respiratory mucosa following challenge and had lower levels of viral genome in nasopharyngeal and bronchoalveolar lavage fluids. The high level of immunogenicity and protective efficacy induced by these vaccine candidates in nonhuman primates suggest that they hold promise for further development.

Ultrasonic vocalizations in golden hamsters (Mesocricetus auratus) reveal modest sex differences and nonlinear signals of sexual motivation.

Vocal signaling is one of many behaviors that animals perform during social interactions. Vocalizations produced by both sexes before mating can communicate sex, identity and condition of the caller. Adult golden hamsters produce ultrasonic vocalizations (USV) after intersexual contact. To determine whether these vocalizations are sexually dimorphic, we analyzed the vocal repertoire for sex differences in: 1) calling rates, 2) composition (structural complexity, call types and nonlinear phenomena) and 3) acoustic structure. In addition, we examined it for individual variation in the calls. The vocal repertoire was mainly composed of 1-note simple calls and at least half of them presented some degree of deterministic chaos. The prevalence of this nonlinear phenomenon was confirmed by low values of harmonic-to-noise ratio for most calls. We found modest sexual differences between repertoires. Males were more likely than females to produce tonal and less chaotic calls, as well as call types with frequency jumps. Multivariate analysis of the acoustic features of 1-note simple calls revealed significant sex differences in the second axis represented mostly by entropy and bandwidth parameters. Male calls showed lower entropy and inter-quartile bandwidth than female calls. Because the variation of acoustic structure within individuals was higher than among individuals, USV could not be reliably assigned to the correct individual. Interestingly, however, this high variability, augmented by the prevalence of chaos and frequency jumps, could be the result of increased vocal effort. Hamsters motivated to produce high calling rates also produced longer calls of broader bandwidth. Thus, the sex differences found could be the result of different sex preferences but also of a sex difference in calling motivation or condition. We suggest that variable and complex USV may have been selected to increase responsiveness of a potential mate by communicating sexual arousal and preventing habituation to the caller.

Reduced mate preference for dominant over subordinate males in old female Syrian hamsters (Mesocricetus auratus).

Why some females choose to mate with a 'preferred' male, whereas others choose to mate with an 'inferior' male is not always clear. Generally, the choosiness of females is thought to decline with advanced age, but relatively few studies have investigated this concept, and reports of this phenomenon in mammals are lacking. To address this deficiency, young and old female golden hamsters were evaluated for their preference for dominant vs. subordinate males. Females observed male dyads as a dominance relationship was established. Dominant and subordinate males were then placed within enclosures at the opposite ends of a Y-maze, and the first approach, scent marking, and time spent near each male were evaluated in young and old females during pro-oestrus-a time when females solicit visits by prospective mates by leaving vaginal and flank scent marks. Whereas the proportion of time spent near the dominant male was significantly greater than random for both young and old females, the proportions of vaginal and flank scent marks left for the dominant male were significantly greater than random for young females, but not for old females. Overall, these results are consistent with a decline in the preference for dominant males by old female hamsters.

A tetravalent alphavirus-vector based dengue vaccine provides effective immunity in an early life mouse model.

Dengue viruses (DENV1-4) cause 390 million clinical infections every year, several hundred thousand of which progress to severe hemorrhagic and shock syndromes. Preexisting immunity resulting from a previous DENV infection is the major risk factor for severe dengue during secondary heterologous infections. During primary infections in infants, maternal antibodies pose an analogous risk. At the same time, maternal antibodies are likely to prevent induction of endogenous anti-DENV antibodies in response to current live, attenuated virus (LAV) vaccine candidates. Any effective early life dengue vaccine has to overcome maternal antibody interference (leading to ineffective vaccination) and poor induction of antibody responses (increasing the risk of severe dengue disease upon primary infection). In a previous study, we demonstrated that a non-propagating Venezuelan equine encephalitis virus replicon expression vector (VRP), expressing the ectodomain of DENV E protein (E85), overcomes maternal interference in a BALB/c mouse model. We report here that a single immunization with a tetravalent VRP vaccine induced NAb and T-cell responses to each serotype at a level equivalent to the monovalent vaccine components, suggesting that this vaccine modality can overcome serotype interference. Furthermore, neonatal immunization was durable and could be boosted later in life to further increase NAb and T-cell responses. Although the neonatal immune response was lower in magnitude than responses in adult BALB/c mice, we demonstrate that VRP vaccines generated protective immunity from a lethal challenge after a single neonatal immunization. In summary, VRP vaccines expressing DENV antigens were immunogenic and protective in neonates, and hence are promising candidates for safe and effective vaccination in early life.

An alphavirus-based adjuvant enhances serum and mucosal antibodies, T cells, and protective immunity to influenza virus in neonatal mice.

UNLABELLED: Neonatal immune responses to infection and vaccination are biased toward TH2 at the cost of proinflammatory TH1 responses needed to combat intracellular pathogens. However, upon appropriate stimulation, the neonatal immune system can induce adult-like TH1 responses. Here we report that a new class of vaccine adjuvant is especially well suited to enhance early life immunity. The GVI3000 adjuvant is a safe, nonpropagating, truncated derivative of Venezuelan equine encephalitis virus that targets dendritic cells (DCs) in the draining lymph node (DLN) and produces intracellular viral RNA without propagating to other cells. RNA synthesis strongly activates the innate immune response so that in adult animals, codelivery of soluble protein antigens induces robust humoral, cellular, and mucosal responses. The adjuvant properties of GVI3000 were tested in a neonatal BALB/c mouse model using inactivated influenza virus (iFlu). After a single immunization, mice immunized with iFlu with the GVI3000 adjuvant (GVI3000-adjuvanted iFlu) had significantly higher and sustained influenza virus-specific IgG antibodies, mainly IgG2a (TH1), compared to the mice immunized with antigen only. GVI3000 significantly increased antigen-specific CD4(+) and CD8(+) T cells, primed mucosal immune responses, and enhanced protection from lethal challenge. As seen in adult mice, the GVI3000 adjuvant increased the DC population in the DLNs, caused activation and maturation of DCs, and induced proinflammatory cytokines and chemokines in the DLNs soon after immunization, including gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), granulocyte colony-stimulating factor (G-CSF), and interleukin 6 (IL-6). In summary, the GVI3000 adjuvant induced an adult-like adjuvant effect with an influenza vaccine and has the potential to improve the immunogenicity and protective efficacy of new and existing neonatal vaccines. IMPORTANCE: The suboptimal immune responses in early life constitute a significant challenge for vaccine design. Here we report that a new class of adjuvant is safe and effective for early life immunization and demonstrate its ability to significantly improve the protective efficacy of an inactivated influenza virus vaccine in a neonatal mouse model. The GVI3000 adjuvant delivers a truncated, self-replicating viral RNA into dendritic cells in the draining lymph node. Intracellular RNA replication activates a strong innate immune response that significantly enhances adaptive antibody and cellular immune responses to codelivered antigens. A significant increase in protection results from a single immunization. Importantly, this adjuvant also primed a mucosal IgA response, which is likely to be critical for protection during many early life infections.

A mucosal adjuvant for the inactivated poliovirus vaccine.

The eradication of poliovirus from the majority of the world has been achieved through the use of two vaccines: the inactivated poliovirus vaccine (IPV) and the live-attenuated oral poliovirus vaccine (OPV). Both vaccines are effective at preventing paralytic poliomyelitis, however, they also have significant differences. Most importantly for this work is the risk of revertant virus from OPV, the greater cost of IPV, and the low mucosal immunity induced by IPV. We and others have previously described the use of an alphavirus-based adjuvant that can induce a mucosal immune response to a co-administered antigen even when delivered at a non-mucosal site. In this report, we describe the use of an alphavirus-based adjuvant (GVI3000) with IPV. The IPV-GVI3000 vaccine significantly increased systemic IgG, mucosal IgG and mucosal IgA antibody responses to all three poliovirus serotypes in mice even when administered intramuscularly. Furthermore, GVI3000 significantly increased the potency of IPV in rat potency tests as measured by poliovirus neutralizing antibodies in serum. Thus, an IPV-GVI3000 vaccine would reduce the dose of IPV needed and provide significantly improved mucosal immunity. This vaccine could be an effective tool to use in the poliovirus eradication campaign without risking the re-introduction of revertant poliovirus derived from OPV.

Role of humoral versus cellular responses induced by a protective dengue vaccine candidate.

With 2.5 billion people at risk, dengue is a major emerging disease threat and an escalating public health problem worldwide. Dengue virus causes disease ranging from a self-limiting febrile illness (dengue fever) to the potentially fatal dengue hemorrhagic fever/dengue shock syndrome. Severe dengue disease is associated with sub-protective levels of antibody, which exacerbate disease upon re-infection. A dengue vaccine should generate protective immunity without increasing severity of disease. To date, the determinants of vaccine-mediated protection against dengue remain unclear, and additional correlates of protection are urgently needed. Here, mice were immunized with viral replicon particles expressing the dengue envelope protein ectodomain to assess the relative contribution of humoral versus cellular immunity to protection. Vaccination with viral replicon particles provided robust protection against dengue challenge. Vaccine-induced humoral responses had the potential to either protect from or exacerbate dengue disease upon challenge, whereas cellular immune responses were beneficial. This study explores the immunological basis of protection induced by a dengue vaccine and suggests that a safe and efficient vaccine against dengue should trigger both arms of the immune system.

Interactions with heterospecific males do not affect how female Mesocricetus hamsters respond to conspecific males.

Reproductive interference includes any interspecific interaction that reduces the fitness of one or both species involved. There are several types of reproductive interference, but they normally involve the direct cost of interacting or mating with heterospecifics. An indirect cost of interacting with heterospecific individuals is a consequent reduction in successful interactions with conspecifics. We tested the hypothesis that being aggressive towards a heterospecific individual will diminish sexual responses towards conspecifics in later encounters. We used two species of Mesocricetus hamsters (Syrian and Turkish hamsters), whose interspecific interactions have previously been determined. We exposed or both exposed and paired Syrian hamster females with a conspecific or a heterospecific male. Five minutes later, we paired all females with a conspecific male and measured the latency to lordosis, the duration of lordosis and any incidence of aggression. We found that (1) interactions with heterospecific males did not affect how females responded to conspecific males in later encounters and (2) previous pairing of female subjects with either conspecific or heterospecific males promoted a faster sexual response by females in subsequent interactions with conspecific males. Thus, aggressive interactions of Syrian hamster females with heterospecific males, contrary to our initial hypothesis, had a positive effect on subsequent interactions with conspecific males.

An alphavirus vector-based tetravalent dengue vaccine induces a rapid and protective immune response in macaques that differs qualitatively from immunity induced by live virus infection.

Despite many years of research, a dengue vaccine is not available, and the more advanced live attenuated vaccine candidate in clinical trials requires multiple immunizations with long interdose periods and provides low protective efficacy. Here, we report important contributions to the development of a second-generation dengue vaccine. First, we demonstrate that a nonpropagating vaccine vector based on Venezuelan equine encephalitis virus replicon particles (VRP) expressing two configurations of dengue virus E antigen (subviral particles [prME] and soluble E dimers [E85]) successfully immunized and protected macaques against dengue virus, while antivector antibodies did not interfere with a booster immunization. Second, compared to prME-VRP, E85-VRP induced neutralizing antibodies faster, to higher titers, and with improved protective efficacy. Third, this study is the first to map antigenic domains and specificities targeted by vaccination versus natural infection, revealing that, unlike prME-VRP and live virus, E85-VRP induced only serotype-specific antibodies, which predominantly targeted EDIII, suggesting a protective mechanism different from that induced by live virus and possibly live attenuated vaccines. Fourth, a tetravalent E85-VRP dengue vaccine induced a simultaneous and protective response to all 4 serotypes after 2 doses given 6 weeks apart. Balanced responses and protection in macaques provided further support for exploring the immunogenicity and safety of this vaccine candidate in humans.

Asymmetric learning to avoid heterospecific males in Mesocricetus hamsters.

If a female mates with a male of a closely related species, her fitness is likely to decline. Consequently, females may develop behavioral mechanisms to avoid mating with heterospecific males. In some species, one such mechanism is for adult females to learn to discriminate against heterospecific males after exposure to such males. We have previously shown that adult, female Syrian hamsters (Mesocricetus auratus) learn to discriminate against male Turkish hamsters (Mesocricetus brandti) after exposure to a single heterospecific male during 8 days across a wire-mesh barrier. Here we repeated that experiment but this time we exposed female Turkish hamsters to a male Syrian hamster for 8 days and then measured sexual and aggressive behaviors towards that heterospecific male and towards a conspecific male. In contrast to female Syrian hamsters, female Turkish hamsters did not differ in their latency to go into lordosis or in any measure of aggression towards either type of male. Female Turkish hamsters spent less time in lordosis with the heterospecific male, but the percentage of trials in which females copulated with conspecific and heterospecific males did not differ. When comparing females from both species that had been exposed to a heterospecific male for 8days, female Syrian hamsters copulated less and were more aggressive towards the heterospecific male compared to the behavior of female Turkish hamsters. We discuss how this asymmetric response between females of the two species may be due to the much larger geographical range of Turkish hamsters compared to Syrian hamsters.

Infected dendritic cells are sufficient to mediate the adjuvant activity generated by Venezuelan equine encephalitis virus replicon particles.

Replicon particles derived from Venezuelan equine encephalitis virus (VEE) are infectious non-propagating particles which act as a safe and potent systemic, mucosal, and cellular adjuvant when delivered with antigen. VEE and VEE replicon particles (VRP) can target multiple cell types including dendritic cells (DCs). The role of these cell types in VRP adjuvant activity has not been previously evaluated, and for these studies we focused on the contribution of DCs to the response to VRP. By analysis of VRP targeting in the draining lymph node, we found that VRP induced rapid recruitment of TNF-secreting monocyte-derived inflammatory dendritic cells. VRP preferentially infected these inflammatory DCs as well as classical DCs and macrophages, with less efficient infection of other cell types. DC depletion suggested that the interaction of VRP with classical DCs was required for recruitment of inflammatory DCs, induction of high levels of many cytokines, and for stable transport of VRP to the draining lymph node. Additionally, in vitro-infected DCs enhanced antigen-specific responses by CD4 and CD8 T cells. By transfer of VRP-infected DCs into mice we showed that these DCs generated an inflammatory state in the draining lymph node similar to that achieved by VRP injection. Most importantly, VRP-infected DCs were sufficient to establish robust adjuvant activity in mice comparable to that produced by VRP injection. These findings indicate that VRP infect, recruit and activate both classical and inflammatory DCs, and those DCs become mediators of the VRP adjuvant activity.

Early activation of the host complement system is required to restrict central nervous system invasion and limit neuropathology during Venezuelan equine encephalitis virus infection.

Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne RNA virus of the genus Alphavirus, family Togaviridae, that is responsible for sporadic outbreaks in human and equid populations in Central and South America. In order to ascertain the role that complement plays in resolving VEEV-induced disease, complement-deficient C3(-/-) mice were infected with a VEEV mutant (V3533) that caused mild, transient disease in immunocompetent mice. In the absence of a functional complement system, peripheral inoculation with V3533 induced much more severe encephalitis. This enhanced pathology was associated with a delay in clearance of infectious virus from the serum and more rapid invasion of the central nervous system in C3(-/-) mice. If V3533 was inoculated directly into the brain, however, disease outcome in C3(-/-) and wild-type mice was identical. These findings indicate that complement-dependent enhancement of peripheral virus clearance is critical for protecting against the development of severe VEEV-induced encephalitis.

A novel self-replicating chimeric lentivirus-like particle.

Successful live attenuated vaccines mimic natural exposure to pathogens without causing disease and have been successful against several viruses. However, safety concerns prevent the development of attenuated human immunodeficiency virus (HIV) as a vaccine candidate. If a safe, replicating virus vaccine could be developed, it might have the potential to offer significant protection against HIV infection and disease. Described here is the development of a novel self-replicating chimeric virus vaccine candidate that is designed to provide natural exposure to a lentivirus-like particle and to incorporate the properties of a live attenuated virus vaccine without the inherent safety issues associated with attenuated lentiviruses. The genome from the alphavirus Venezuelan equine encephalitis virus (VEE) was modified to express SHIV89.6P genes encoding the structural proteins Gag and Env. Expression of Gag and Env from VEE RNA in primate cells led to the assembly of particles that morphologically and functionally resembled lentivirus virions and that incorporated alphavirus RNA. Infection of CD4⁺ cells with chimeric lentivirus-like particles was specific and productive, resulting in RNA replication, expression of Gag and Env, and generation of progeny chimeric particles. Further genome modifications designed to enhance encapsidation of the chimeric virus genome and to express an attenuated simian immunodeficiency virus (SIV) protease for particle maturation improved the ability of chimeric lentivirus-like particles to propagate in cell culture. This study provides proof of concept for the feasibility of creating chimeric virus genomes that express lentivirus structural proteins and assemble into infectious particles for presentation of lentivirus immunogens in their native and functional conformation.

The role of the blood-brain barrier during Venezuelan equine encephalitis virus infection.

Venezuelan equine encephalitis (VEE) virus is a mosquito-borne alphavirus associated with sporadic outbreaks in human and equid populations in the Western Hemisphere. After the bite of an infected mosquito, the virus initiates a biphasic disease: a peripheral phase with viral replication in lymphoid and myeloid tissues, followed by a neurotropic phase with infection of central nervous system (CNS) neurons, causing neuropathology and in some cases fatal encephalitis. The mechanisms allowing VEE virus to enter the CNS are currently poorly understood. Previous data have shown that the virus gains access to the CNS by infecting olfactory sensory neurons in the nasal mucosa of mice. However, at day 5 after inoculation, the infection of the brain is multifocal, indicating that virus particles are able to cross the blood-brain barrier (BBB). To better understand the role of the BBB during VEE virus infection, we used a well-characterized mouse model system. Using VEE virus replicon particles (VRP), we modeled the early events of neuroinvasion, showing that the replication of VRP in the nasal mucosa induced the opening of the BBB, allowing peripherally administered VRP to invade the brain. Peripheral VEE virus infection was characterized by a biphasic opening of the BBB. Further, inhibition of BBB opening resulted in a delayed viral neuroinvasion and pathogenesis. Overall, these results suggest that VEE virus initially enters the CNS through the olfactory pathways and initiates viral replication in the brain, which induces the opening of the BBB, allowing a second wave of invading virus from the periphery to enter the brain.

Adult female hamsters require long and sustained exposures to heterospecific males to avoid interspecific mating.

Interspecific mating normally decreases female fitness. In many species, females avoid heterospecific males innately or by imprinting on their parents. Alternatively, adult females could learn to discriminate against heterospecific males after exposure to such males. For example, Syrian hamster (Mesocricetus auratus) females learn to discriminate between conspecific males and Turkish hamster (M. brandti) males during adulthood by exposure to males of both species. Adult females not previously exposed to Turkish hamster males will mate similarly with conspecific and heterospecific males. However, in a previous study we showed that exposure to a heterospecific male and a conspecific male for 8 days led to mating avoidance and aggression towards the heterospecific male. Here we conducted two experiments to investigate how much exposure to the heterospecific male was required for females to avoid mating with the heterospecific male (Experiment 1) and how long that avoidance lasted in the absence of continuous exposure to heterospecific stimuli (Experiment 2). Fast and durable learning would indicate the evolution of an efficient avoidance response. In Experiment 1, females were exposed to a heterospecific male for 1, 4 h, 4 or 8 days and then paired with that male. We found more avoidance of interspecific mating after 4 or 8 days of exposure than after 1 or 4 h of exposure. In Experiment 2, females were exposed to a heterospecific male for 8 days and then paired with that male either 10 min later or 8 days later. We found that after an 8-day delay females were highly sexually receptive to the heterospecific male. Additionally, a comparison between the current experiments and a previous study indicates that female Syrian hamsters do not require concurrent exposure to a conspecific male and a heterospecific male to learn to avoid interspecific mating; exposure to a heterospecific male is sufficient.

Avoidance of interspecific mating in female Syrian hamsters is stronger toward familiar than toward unfamiliar heterospecific males.

Adult Syrian hamster females (Mesocricetus auratus) learn to discriminate against familiar heterospecific males (Turkish hamster, M. brandti). We investigated whether females learn to avoid any heterospecific male after exposure to just one heterospecific male. We predicted that, after being exposed to one heterospecific male, a female would avoid mating not only with that familiar male but also with any unfamiliar heterospecific male. We exposed females to a heterospecific male across a wire-mesh barrier for 8 days and then paired the female with (a) that same heterospecific male or (b) an unfamiliar heterospecific male. Females exhibited lordosis faster and for a longer duration toward the unfamiliar than toward the familiar heterospecific male. However, females were similarly aggressive toward familiar and unfamiliar heterospecific males. Perhaps exposure to stimuli from several heterospecific males (a likely scenario in the wild) would result in females behaving similarly toward familiar and unfamiliar heterospecific males.

Alphavirus replicon-based adjuvants enhance the immunogenicity and effectiveness of Fluzone ® in rhesus macaques.

Venezuelan equine encephalitis virus replicon particles (VRP) without a transgene (null VRP) have been used to adjuvant effective humoral [1], cellular [2], and mucosal [3] immune responses in mice. To assess the adjuvant activity of null VRP in the context of a licensed inactivated influenza virus vaccine, rhesus monkeys were immunized with Fluzone(®) alone or Fluzone(®) mixed with null VRP and then challenged with a human seasonal influenza isolate, A/Memphis/7/2001 (H1N1). Compared to Fluzone(®) alone, Fluzone(®)+null VRP immunized animals had stronger influenza-specific CD4(+) T cell responses (4.4 fold) with significantly higher levels of virus-specific IFN-γ (7.6 fold) and IL-2 (5.3 fold) producing CD4+ T cells. Fluzone(®)+null VRP immunized animals also had significantly higher plasma anti-influenza IgG (p<0.0001, 1.3 log) and IgA (p<0.05, 1.2 log) levels. In fact, the mean plasma anti-influenza IgG titers after one Fluzone(®)+null VRP immunization was 1.2 log greater (p<0.04) than after two immunizations with Fluzone(®) alone. After virus challenge, only Fluzone(®)+null VRP immunized monkeys had a significantly lower level of viral replication (p<0.001) relative to the unimmunized control animals. Although little anti-influenza antibody was detected in the respiratory secretions after immunization, strong anamnestic anti-influenza IgG and IgA responses were present in secretions of the Fluzone(®)+null VRP immunized monkeys immediately after challenge. There were significant inverse correlations between influenza RNA levels in tracheal lavages and plasma anti-influenza HI and IgG anti-influenza antibody titers prior to challenge. These results demonstrate that null VRP dramatically improve both the immunogenicity and protection elicited by a licensed inactivated influenza vaccine.

Syrian hamster males below an age threshold do not elicit aggression from unfamiliar adult males.

In many species, young males are the dispersers, leaving their natal area after weaning to establish a breeding area of their own. As young males disperse, however, they are bound to encounter unfamiliar adult males with established territories. Such interactions between an adult male and a young male may always be agonistic. Alternatively, there may be an age threshold below which aggression is not elicited and above which the adult male is aggressive toward the juvenile male. To test these two alternative hypotheses, we paired 47 young Syrian hamster (Mesocricetus auratus) males ranging from 24 to 65 days of age with 47 adult male hamsters and measured aggressive and investigatory behavior for 5 min. We observed no aggression by the adult toward young males between 24 and 47 days of age or toward the single male that was 49 days of age. Young males that were 50 days of age or older, however, elicited significant levels of aggression from the adults. These results indicate that in Syrian hamsters, young males are less vulnerable to adult aggression up to an age threshold and are more vulnerable to adult aggression beyond that threshold. This pattern may facilitate the establishment of territories by dispersing young males below that age threshold.

Adult female hamsters avoid interspecific mating after exposure to heterospecific males.

When females mate with a heterospecific male, they do not usually produce viable offspring. Thus, there is a selective pressure for females to avoid interspecific mating. In many species, females innately avoid heterospecific males; females can also imprint on their parents to avoid later sexual interactions with heterospecific males. However, it was previously unknown whether adult females can learn to discriminate against heterospecific males. We tested the hypothesis that adult females previously unable to avoid interspecific mating learn to avoid such mating after being exposed to heterospecific males. Syrian hamster (Mesocricetus auratus) females not previously exposed to Turkish hamster (Mesocricetus brandti) males can discriminate between odors of conspecific and heterospecific males, but they mate with either type of male. However, when we exposed adult females to both a conspecific male and a heterospecific male through wire-mesh barriers for 8 days, and then paired them sequentially with the two males, females were more receptive to conspecific males and more aggressive to heterospecific males. When females were paired with the heterospecific male first and the conspecific male second, no female was receptive and all were aggressive to heterospecific males. When females were paired with the conspecific male first, only 43% of females were then aggressive toward the heterospecific male. That is, interactions with conspecific males may decrease a female's ability to properly avoid heterospecific males. Our study clearly shows for the first time that females can learn during adulthood to avoid interspecific mating just by being exposed to stimuli from heterospecific males.