Impaired immune response to vaccination against infection with human respiratory syncytial virus at advanced age.

UNLABELLED: Elderly humans are prone to severe infection with human respiratory syncytial virus (HRSV). The aging of today's human population warrants the development of protective vaccination strategies aimed specifically at the elderly. This may require special approaches due to deteriorating immune function. To design and test vaccination strategies tailored to the elderly population, we need to understand the host response to HRSV vaccination and infection at old age. Moreover, the preclinical need for testing of candidate vaccines requires translational models resembling susceptibility to the (unadapted) human pathogen. Here, we explored the effects of aging on immunity and protection induced by a model HRSV vaccine candidate in a translational aging model in cotton rats (Sigmodon hispidus) and examined possibilities to optimize vaccination concepts for the elderly. We immunized young and aged cotton rats with a live-attenuated recombinant HRSV vaccine candidate and analyzed the induced immune response to and protection against challenge with HRSV. In old cotton rats, HRSV infection persisted longer, and vaccination induced less protection against infection. Aged animals developed lower levels of vaccine-induced IgG, virus-neutralizing serum antibodies, and IgA in lungs. Moreover, booster responses to HRSV challenge were impaired in animals vaccinated at an older age. However, increased dose and reduced attenuation of vaccine improved protection even in old animals. This study shows that cotton rats provide a model for studying the effects of aging on the immune response to the human respiratory pathogen HRSV and possibilities to optimize vaccine concepts for the elderly. IMPORTANCE: HRSV infection poses a risk for severe disease in the elderly. The aging of the population warrants increased efforts to prevent disease at old age, whereas HRSV vaccines are only in the developmental phase. The preclinical need for testing of candidate human vaccines requires translational models resembling susceptibility to the natural human virus. Moreover, we need to gain insight into waning immunity at old age, as this is a special concern in vaccine development. In this study, we explored the effect of age on protection and immunity against an experimental HRSV vaccine in aged cotton rats (Sigmodon hispidus), a rodent species that provides a model representing natural susceptibility to human viruses. Older animals generate fewer antibodies upon vaccination and require a higher vaccine dose for protection. Notably, during the early secondary immune response to subsequent HRSV infection, older animals showed less protection and a slower increase of the virus-neutralizing antibody titer.

Infection-enhancing lipopeptides do not improve intranasal immunization of cotton rats with a delta-G candidate live-attenuated human respiratory syncytial virus vaccine.

Development of live-attenuated human respiratory syncytial virus (HRSV) vaccines has proven to be difficult. Several vaccine candidates were found to be over-attenuated and displayed limited immunogenicity. Recently, we identified three synthetic cationic lipopeptides that enhanced paramyxovirus infections in vitro. The infection enhancement proved to be mediated by enhanced virus binding to target cells. We hypothesized that these lipopeptides can be used as adjuvants to promote immune responses induced by live-attenuated paramyxovirus vaccines. This hypothesis was tested in a vaccination and challenge model in cotton rats, using a previously described recombinant live-attenuated candidate HRSV vaccine lacking the gene encoding the G glycoprotein (rHRSVΔG). Surprisingly, intranasal vaccination of cotton rats with rHRSVΔG formulated in infection-enhancing lipopeptides resulted in reduced virus loads in nasopharyngeal lavages, reduced seroconversion levels and reduced protection from wild-type HRSV challenge. In conclusion, we were unable to demonstrate the feasibility of lipopeptides as adjuvants for a candidate live-attenuated HRSV vaccine in the cotton rat model.

An improved respiratory syncytial virus neutralization assay based on the detection of green fluorescent protein expression and automated plaque counting.

BACKGROUND: Virus neutralizing antibodies against respiratory syncytial virus (RSV) are considered important correlates of protection for vaccine evaluation. The established plaque reduction assay is time consuming, labor intensive and highly variable. METHODS: Here, a neutralization assay based on a modified RSV strain expressing the green fluorescent protein in combination with automated detection and quantification of plaques is described. RESULTS: The fluorescence plaque reduction assay in microplate format requires only two days to complete and is simple and reproducible. A good correlation between visual and automated counting methods to determine RSV neutralizing serum antibody titers was observed. CONCLUSIONS: The developed virus neutralization assay is suitable for high-throughput testing and can be used for both animal studies and (large scale) vaccine clinical trials.

Genome-wide association study identifies single nucleotide polymorphism in DYRK1A associated with replication of HIV-1 in monocyte-derived macrophages.

BACKGROUND: HIV-1 infected macrophages play an important role in rendering resting T cells permissive for infection, in spreading HIV-1 to T cells, and in the pathogenesis of AIDS dementia. During highly active anti-retroviral treatment (HAART), macrophages keep producing virus because tissue penetration of antiretrovirals is suboptimal and the efficacy of some is reduced. Thus, to cure HIV-1 infection with antiretrovirals we will also need to efficiently inhibit viral replication in macrophages. The majority of the current drugs block the action of viral enzymes, whereas there is an abundance of yet unidentified host factors that could be targeted. We here present results from a genome-wide association study identifying novel genetic polymorphisms that affect in vitro HIV-1 replication in macrophages. METHODOLOGY/PRINCIPAL FINDINGS: Monocyte-derived macrophages from 393 blood donors were infected with HIV-1 and viral replication was determined using Gag p24 antigen levels. Genomic DNA from individuals with macrophages that had relatively low (n = 96) or high (n = 96) p24 production was used for SNP genotyping with the Illumina 610 Quad beadchip. A total of 494,656 SNPs that passed quality control were tested for association with HIV-1 replication in macrophages, using linear regression. We found a strong association between in vitro HIV-1 replication in monocyte-derived macrophages and SNP rs12483205 in DYRK1A (p = 2.16 × 10(-5)). While the association was not genome-wide significant (p<1 × 10(-7)), we could replicate this association using monocyte-derived macrophages from an independent group of 31 individuals (p = 0.0034). Combined analysis of the initial and replication cohort increased the strength of the association (p = 4.84 × 10(-6)). In addition, we found this SNP to be associated with HIV-1 disease progression in vivo in two independent cohort studies (p = 0.035 and p = 0.0048). CONCLUSIONS/SIGNIFICANCE: These findings suggest that the kinase DYRK1A is involved in the replication of HIV-1, in vitro in macrophages as well as in vivo.

A highly attenuated recombinant human respiratory syncytial virus lacking the G protein induces long-lasting protection in cotton rats.

BACKGROUND: Respiratory syncytial virus (RSV) is a primary cause of serious lower respiratory tract illness for which there is still no safe and effective vaccine available. Using reverse genetics, recombinant (r)RSV and an rRSV lacking the G gene (DeltaG) were constructed based on a clinical RSV isolate (strain 98-25147-X). RESULTS: Growth of both recombinant viruses was equivalent to that of wild type virus in Vero cells, but was reduced in human epithelial cells like Hep-2. Replication in cotton rat lungs could not be detected for DeltaG, while rRSV was 100-fold attenuated compared to wild type virus. Upon single dose intranasal administration in cotton rats, both recombinant viruses developed high levels of neutralizing antibodies and conferred comparable long-lasting protection against RSV challenge; protection against replication in the lungs lasted at least 147 days and protection against pulmonary inflammation lasted at least 75 days. CONCLUSION: Collectively, the data indicate that a single dose immunization with the highly attenuated DeltaG as well as the attenuated rRSV conferred long term protection in the cotton rat against subsequent RSV challenge, without inducing vaccine enhanced pathology. Since DeltaG is not likely to revert to a less attenuated phenotype, we plan to evaluate this deletion mutant further and to investigate its potential as a vaccine candidate against RSV infection.

Donor variation in in vitro HIV-1 susceptibility of monocyte-derived macrophages.

Primary human cells from different donors vary in their susceptibility to in vitro infection with HIV-1. In order to perform genetic analysis to identify host factors that affect HIV-1 susceptibility, it is important that a clear phenotype is defined. Here, we report a standardized method to study variation for in vitro HIV-1 infection in monocyte-derived macrophages (MDM) from large numbers of individuals. With this assay, HIV-1 susceptibility of MDM from 489 different donors shows more than 3 log variation and a good correlation with the 32 base pair deletion in the CCR5 co-receptor (ccr5 Delta 32 genotype) of the donors. However, in 7 of 12 donors completely resistant to infection with CCR5-using HIV-1, this was not explained by the ccr5 Delta 32 genotype, showing evidence that other host factors are likely to influence HIV-1 replication in MDM. Infections with VSV-G pseudotyped HIV-1 indeed confirmed the existence of post-entry level restrictions in MDM.

Identification, phenotypic analysis, and full-length nucleotide sequence of a Dutch Coxsackievirus A20 isolate.

Recombination between (human) enteroviruses is a common event in nature. Recently, it has been recognised that this feature has a major impact on the use of the live-attenuated polio vaccine during the end stage of polio eradication. The constraints for successful recombination between (vaccine-derived) polioviruses and human enteroviruses are, however, largely unknown. Here, we describe the identification and characterisation of a HEV-C field strain, isolated from the stool of a 2-year-old Dutch boy. Serotyping indicated that strain 89490 is a variant of strain CAV20a, which is already a variant of the prototype CAV20 strain. Amino acid sequence identity of 94.1% for the entire P1-region, and 92.4% for the major structural protein (VP1) indicates that this strain is indeed related to CAV20. However, virus neutralisation and Western blot analysis failed to show antigenic homology between the prototype CAV20 strain and our field strain. Furthermore, the 89490 field strain, just like the sub-prototype CAV20a, is able to replicate on RD-cells, while the prototype CAV20 and another sub-prototype CAV20b are not. On the basis of the phylogenetic analysis of the P2 and P3 region we expect that strain 89490 can act as recombination partner for the attenuated poliovirus strains of the Oral Polio Vaccine (OPV).