Development of Virus-like Particle Plant-Based Vaccines against Avian H5 and H9 Influenza A Viruses.

Avian influenza A virus (AIV) is a significant cause of mortality in poultry, causing substantial economic loss, particularly in developing countries, and has zoonotic potential. For example, highly pathogenic avian influenza (HPAI) viruses of the H5 subtype have been circulating in Egypt for around two decades. In the last decade, H5N1 viruses of clade 2.2.1 have been succeeded by the antigenically distinct H5N8 clade 2.3.4.4b viruses. Furthermore, H9N2 viruses co-circulate with the H5N8 viruses in Egyptian poultry. It is widely recognised that effective vaccination against IAV requires a close antigenic match between the vaccine and viruses circulating in the field. Therefore, approaches to develop cost-effective vaccines that can be rapidly adapted to local virus strains are required for developing countries such as Egypt. In this project, the haemagglutinin (HA) proteins of Egyptian H5 and H9 viruses were expressed by transient transfection of plants (Nicotiana benthamiana). The formation of virus-like particles (VLPs) was confirmed by transmission electron microscopy. Mice were immunised with four doses of either H5 or H9 VLPs with adjuvant. Antibody and cellular immune responses were measured against the corresponding recombinant protein using ELISA and enzyme-linked immunosorbent assay (ELISpot), respectively. Chickens were immunised with one dose of H5 VLPs, eliciting HA-specific antibodies measured by ELISA and a pseudotyped virus neutralisation test using a heterologous H5 HA. In conclusion, plant-based VLP vaccines have potential for producing an effective vaccine candidate within a short time at a relatively low cost.

Lipid biophysics and/or soft matter-inspired approach for controlling enveloped virus infectivity.

Proven as a natural barrier against viral infection, pulmonary surfactant phospholipids have a biophysical and immunological role within the respiratory system, acting against microorganisms including viruses. Enveloped viruses have, in common, an outer bilayer membrane that forms the underlying structure for viral membrane proteins to function in an optimal way to ensure infectivity. Perturbating the membrane of viruses using exogenous lipids can be envisioned as a generic way to reduce their infectivity. In this context, the potential of exogenous lipids to be used against enveloped virus infectivity would be indicated by the resulting physical stress imposed to the viral membrane, and conical lipids, i.e. lyso-lipids, would be expected to generate stronger biophysical disturbances. We confirm that when treated with lyso-lipids the infectivity three strains of influenza virus (avian H2N3, equine H3N8 or pandemic human influenza H1N1) is reduced by up to 99% in a cell-based model. By contrast, lipids with a similar head group but two aliphatic chains were less effective (reducing infection by only 40-50%). This work opens a new path to merge concepts from different research fields, i.e. 'soft matter physics' and virology.

Recombinant Viruses for Cancer Therapy.

Recombinant viruses are novel therapeutic agents that can be utilized for treatment of various diseases, including cancers. Recombinant viruses can be engineered to express foreign transgenes and have a broad tropism allowing gene expression in a wide range of host cells. They can be selected or designed for specific therapeutic goals; for example, recombinant viruses could be used to stimulate host immune response against tumor-specific antigens and therefore overcome the ability of the tumor to evade the host's immune surveillance. Alternatively, recombinant viruses could express immunomodulatory genes which stimulate an anti-cancer immune response. Oncolytic viruses can replicate specifically in tumor cells and induce toxic effects leading to cell lysis and apoptosis. However, each of these approaches face certain difficulties that must be resolved to achieve maximum therapeutic efficacy. In this review we discuss actively developing approaches for cancer therapy based on recombinant viruses, problems that need to be overcome, and possible prospects for further development of recombinant virus based therapy.

DNA microarray global gene expression analysis of influenza virus-infected chicken and duck cells.

The data described in this article pertain to the article by Kuchipudi et al. (2014) titled "Highly Pathogenic Avian Influenza Virus Infection in Chickens But Not Ducks Is Associated with Elevated Host Immune and Pro-inflammatory Responses" [1]. While infection of chickens with highly pathogenic avian influenza (HPAI) H5N1 virus subtypes often leads to 100% mortality within 1 to 2 days, infection of ducks in contrast causes mild or no clinical signs. The rapid onset of fatal disease in chickens, but with no evidence of severe clinical symptoms in ducks, suggests underlying differences in their innate immune mechanisms. We used Chicken Genechip microarrays (Affymetrix) to analyse the gene expression profiles of primary chicken and duck lung cells infected with a low pathogenic avian influenza (LPAI) H2N3 virus and two HPAI H5N1 virus subtypes to understand the molecular basis of host susceptibility and resistance in chickens and ducks. Here, we described the experimental design, quality control and analysis that were performed on the data set. The data are publicly available through the Gene Expression Omnibus (GEO)database with accession number GSE33389, and the analysis and interpretation of these data are included in Kuchipudi et al. (2014) [1].

Identification of morphological differences between avian influenza A viruses grown in chicken and duck cells.

Although wild ducks are considered to be the major reservoirs for most influenza A virus subtypes, they are typically resistant to the effects of the infection. In contrast, certain influenza viruses may be highly pathogenic in other avian hosts such as chickens and turkeys, causing severe illness and death. Following in vitro infection of chicken and duck embryo fibroblasts (CEF and DEF) with low pathogenic avian influenza (LPAI) viruses, duck cells die more rapidly and produce fewer infectious virions than chicken cells. In the current study, the morphology of viruses produced from CEF and DEF cells infected with low pathogenic avian H2N3 was examined. Transmission electron microscopy showed that viruses budding from duck cells were elongated, while chicken cells produced mostly spherical virions; similar differences were observed in viral supernatants. Sequencing of the influenza genome of chicken- and duck-derived H2N3 LPAI revealed no differences, implicating host cell determinants as responsible for differences in virus morphology. Both DEF and CEF cells produced filamentous virions of equine H3N8 (where virus morphology is determined by the matrix gene). DEF cells produced filamentous or short filament virions of equine H3N8 and avian H2N3, respectively, even after actin disruption with cytochalasin D. These findings suggest that cellular factors other than actin are responsible for the formation of filamentous virions in DEF cells. The formation of elongated virions in duck cells may account for the reduced number of infectious virions produced and could have implications for virus transmission or maintenance in the reservoir host.

Highly pathogenic avian influenza virus infection in chickens but not ducks is associated with elevated host immune and pro-inflammatory responses.

Highly pathogenic avian influenza (HPAI) H5N1 viruses cause severe infection in chickens at near complete mortality, but corresponding infection in ducks is typically mild or asymptomatic. To understand the underlying molecular differences in host response, primary chicken and duck lung cells, infected with two HPAI H5N1 viruses and a low pathogenicity avian influenza (LPAI) H2N3 virus, were subjected to RNA expression profiling. Chicken cells but not duck cells showed highly elevated immune and pro-inflammatory responses following HPAI virus infection. HPAI H5N1 virus challenge studies in chickens and ducks corroborated the in vitro findings. To try to determine the underlying mechanisms, we investigated the role of signal transducer and activator of transcription-3 (STAT-3) in mediating pro-inflammatory response to HPAIV infection in chicken and duck cells. We found that STAT-3 expression was down-regulated in chickens but was up-regulated or unaffected in ducks in vitro and in vivo following H5N1 virus infection. Low basal STAT-3 expression in chicken cells was completely inhibited by H5N1 virus infection. By contrast, constitutively active STAT-3 detected in duck cells was unaffected by H5N1 virus infection. Transient constitutively-active STAT-3 transfection in chicken cells significantly reduced pro-inflammatory response to H5N1 virus infection; on the other hand, chemical inhibition of STAT-3 activation in duck cells increased pro-inflammatory gene expression following H5N1 virus infection. Collectively, we propose that elevated pro-inflammatory response in chickens is a major pathogenicity factor of HPAI H5N1 virus infection, mediated in part by the inhibition of STAT-3.

A consensus approach to vertebrate de novo transcriptome assembly from RNA-seq data: assembly of the duck (Anas platyrhynchos) transcriptome.

For vertebrate organisms where a reference genome is not available, de novo transcriptome assembly enables a cost effective insight into the identification of tissue specific or differentially expressed genes and variation of the coding part of the genome. However, since there are a number of different tools and parameters that can be used to reconstruct transcripts, it is difficult to determine an optimal method. Here we suggest a pipeline based on (1) assessing the performance of three different assembly tools (2) using both single and multiple k-mer (MK) approaches (3) examining the influence of the number of reads used in the assembly (4) merging assemblies from different tools. We use an example dataset from the vertebrate Anas platyrhynchos domestica (Pekin duck). We find that taking a subset of data enables a robust assembly to be produced by multiple methods without the need for very high memory capacity. The use of reads mapped back to transcripts (RMBT) and CEGMA (Core Eukaryotic Genes Mapping Approach) provides useful metrics to determine the completeness of assembly obtained. For this dataset the use of MK in the assembly generated a more complete assembly as measured by greater number of RMBT and CEGMA score. Merged single k-mer assemblies are generally smaller but consist of longer transcripts, suggesting an assembly consisting of fewer fragmented transcripts. We suggest that the use of a subset of reads during assembly allows the relatively rapid investigation of assembly characteristics and can guide the user to the most appropriate transcriptome for particular downstream use. Transcriptomes generated by the compared assembly methods and the final merged assembly are freely available for download at http://dx.doi.org/10.6084/m9.figshare.1032613.

18S rRNA is a reliable normalisation gene for real time PCR based on influenza virus infected cells.

BACKGROUND: One requisite of quantitative reverse transcription PCR (qRT-PCR) is to normalise the data with an internal reference gene that is invariant regardless of treatment, such as virus infection. Several studies have found variability in the expression of commonly used housekeeping genes, such as beta-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), under different experimental settings. However, ACTB and GAPDH remain widely used in the studies of host gene response to virus infections, including influenza viruses. To date no detailed study has been described that compares the suitability of commonly used housekeeping genes in influenza virus infections. The present study evaluated several commonly used housekeeping genes [ACTB, GAPDH, 18S ribosomal RNA (18S rRNA), ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide (ATP5B) and ATP synthase, H+ transporting, mitochondrial Fo complex, subunit C1 (subunit 9) (ATP5G1)] to identify the most stably expressed gene in human, pig, chicken and duck cells infected with a range of influenza A virus subtypes. RESULTS: The relative expression stability of commonly used housekeeping genes were determined in primary human bronchial epithelial cells (HBECs), pig tracheal epithelial cells (PTECs), and chicken and duck primary lung-derived cells infected with five influenza A virus subtypes. Analysis of qRT-PCR data from virus and mock infected cells using NormFinder and BestKeeper software programmes found that 18S rRNA was the most stable gene in HBECs, PTECs and avian lung cells. CONCLUSIONS: Based on the presented data from cell culture models (HBECs, PTECs, chicken and duck lung cells) infected with a range of influenza viruses, we found that 18S rRNA is the most stable reference gene for normalising qRT-PCR data. Expression levels of the other housekeeping genes evaluated in this study (including ACTB and GPADH) were highly affected by influenza virus infection and hence are not reliable as reference genes for RNA normalisation.

Mammalian innate resistance to highly pathogenic avian influenza H5N1 virus infection is mediated through reduced proinflammation and infectious virus release.

Respiratory epithelial cells and macrophages are the key innate immune cells that play an important role in the pathogenesis of influenza A virus infection. We found that these two cell types from both human and pig showed comparable susceptibilities to initial infection with a highly pathogenic avian influenza (HPAI) H5N1 virus (A/turkey/Turkey/1/05) and a moderately pathogenic human influenza H1N1 virus (A/USSR/77), but there were contrasting differences in host innate immune responses. Human cells mounted vigorous cytokine (tumor necrosis factor alpha [TNF-α] and interleukin-6 [IL-6]) and chemokine (CXCL9, CXCL10, and CXCL11) responses to H5N1 virus infection. However, pig epithelial cells and macrophages showed weak or no TNF-α and chemokine induction with the same infections. The apparent lack of a strong proinflammatory response, corroborated by the absence of TNF-α induction in H5N1 virus-challenged pigs, coincided with greater cell death and the reduced release of infectious virus from infected pig epithelial cells. Suppressor of cytokine signaling 3 (SOCS3), a protein suppressor of the JAK-STAT pathway, was constitutively highly expressed and transcriptionally upregulated in H5N1 virus-infected pig epithelial cells and macrophages, in contrast to the corresponding human cells. The overexpression of SOCS3 in infected human macrophages dampened TNF-α induction. In summary, we found that the reported low susceptibility of pigs to contemporary Eurasian HPAI H5N1 virus infections coincides at the level of innate immunity of respiratory epithelial cells and macrophages with a reduced output of viable virus and an attenuated proinflammatory response, possibly mediated in part by SOCS3, which could serve as a target in the treatment or prevention of virus-induced hypercytokinemia, as observed for humans.

Rapid death of duck cells infected with influenza: a potential mechanism for host resistance to H5N1.

Aquatic birds are the natural reservoir for most subtypes of influenza A, and a source of novel viruses with the potential to cause human pandemics, fatal zoonotic disease or devastating epizootics in poultry. It is well recognised that waterfowl typically show few clinical signs following influenza A infection, in contrast, terrestrial poultry such as chickens may develop severe disease with rapid death following infection with highly pathogenic avian influenza. This study examined the cellular response to influenza infection in primary cells derived from resistant (duck) and susceptible (chicken) avian hosts. Paradoxically, we observed that duck cells underwent rapid cell death following infection with low pathogenic avian H2N3, classical swine H1N1 and 'classical' highly pathogenic H5N1 viruses. Dying cells showed morphological features of apoptosis, increased DNA fragmentation and activation of caspase 3/7. Following infection of chicken cells, cell death occurred less rapidly, accompanied by reduced DNA fragmentation and caspase activation. Duck cells produced similar levels of viral RNA but less infectious virus, in comparison with chicken cells. Such rapid cell death was not observed in duck cells infected with a contemporary Eurasian lineage H5N1 fatal to ducks. The induction of rapid death in duck cells may be part of a mechanism of host resistance to influenza A, with the loss of this response leading to increased susceptibility to emergent strains of H5N1. These studies provide novel insights that should help resolve the long-standing enigma of host-pathogen relationships for highly pathogenic and zoonotic avian influenza.

Evaluation of cytokine mRNA expression in bronchoalveolar lavage cells from horses with inflammatory airway disease.

Inflammatory airway disease (IAD) is a common disorder of performance horses and is associated with poor performance and accumulation of mucus and inflammatory cells in lower airway secretions. Horses with IAD frequently have increased relative counts of neutrophils in bronchoalveolar lavage fluid (BALF); less commonly relative counts of eosinophils and/or mast cells may be increased. The aetiopathogenesis of IAD is unknown and may involve innate and/or acquired immune responses to various factors including respirable dust constituents, micro-organisms, noxious gases and unconditioned air. The molecular pathways and role of the immune system in the pathogenesis of IAD remain poorly defined and it is unknown whether polarised T cell responses occur in the disease, as have been reported to occur in equine recurrent airway obstruction and asthma in humans. Elucidating cytokine responses that develop in horses with IAD may allow a greater understanding of the possible aetiopathological pathway(s) involved and could contribute to development of novel treatments. We compared the mRNA expression of tumour necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), interleukin (IL)-1ß, IL-2, IL-4, IL-8, IL-13, IL-17 and IL-23 in cell pellets extracted from BALF of horses with IAD (n=21) and horses free of respiratory tract disease (n=17). Horses with IAD had significantly increased levels of TNF-α, IL-1ß and IL-23 mRNA; no significant differences in the other cytokine mRNAs were detected. The results of this study indicate that IAD of horses is associated with increased mRNA expression of pro-inflammatory cytokines in BALF cells, which may reflect stimulation of the innate immune responses to inhaled antigens. There was no evidence of a polarised T-cell cytokine response suggesting hypersensitivity responses may not be involved in the aetiopathogenesis of IAD.

Comparative distribution of human and avian type sialic acid influenza receptors in the pig.

BACKGROUND: A major determinant of influenza infection is the presence of virus receptors on susceptible host cells to which the viral haemagglutinin is able to bind. Avian viruses preferentially bind to sialic acid alpha2,3-galactose (SAalpha2,3-Gal) linked receptors, whereas human strains bind to sialic acid alpha2,6-galactose (SAalpha2,6-Gal) linked receptors. To date, there has been no detailed account published on the distribution of SA receptors in the pig, a model host that is susceptible to avian and human influenza subtypes, thus with potential for virus reassortment. We examined the relative expression and spatial distribution of SAalpha2,3-GalG(1-3)GalNAc and SAalpha2,6-Gal receptors in the major organs from normal post-weaned pigs by binding with lectins Maackia amurensis agglutinins (MAA II) and Sambucus nigra agglutinin (SNA) respectively. RESULTS: Both SAalpha2,3-Gal and SAalpha2,6-Gal receptors were extensively detected in the major porcine organs examined (trachea, lung, liver, kidney, spleen, heart, skeletal muscle, cerebrum, small intestine and colon). Furthermore, distribution of both SA receptors in the pig respiratory tract closely resembled the published data of the human tract. Similar expression patterns of SA receptors between pig and human in other major organs were found, with exception of the intestinal tract. Unlike the limited reports on the scarcity of influenza receptors in human intestines, we found increasing presence of SAalpha2,3-Gal and SAalpha2,6-Gal receptors from duodenum to colon in the pig. CONCLUSIONS: The extensive presence of SAalpha2,3-Gal and SAalpha2,6-Gal receptors in the major organs examined suggests that each major organ may be permissive to influenza virus entry or infection. The high similarity of SA expression patterns between pig and human, in particular in the respiratory tract, suggests that pigs are not more likely to be potential hosts for virus reassortment than humans. Our finding of relative abundance of SA receptors in the pig intestines highlights a need for clarification on the presence of SA receptors in the human intestinal tract.

Retroviral infections of small animals.

Retroviral infections are particularly important in cats, which are commonly infected with feline leukemia virus and feline immunodeficiency virus. This article describes the biology of these viruses and explores current issues regarding vaccination and diagnosis. The seeming lack of a recognized retrovirus infection in dogs is speculated on, and current and potential future therapies are discussed.

Prime-boost vaccination using DNA and whole inactivated virus vaccines provides limited protection against virulent feline immunodeficiency virus.

Protection against feline immunodeficiency virus (FIV) has been achieved using a variety of vaccines notably whole inactivated virus (WIV) and DNA. However protection against more virulent isolates, typical of those encountered in natural infections, has been difficult to achieve. In an attempt to improve protection against virulent FIV(GL8), we combined both DNA and WIV vaccines in a "prime-boost" approach. Thirty cats were divided into four groups receiving vaccinations and one unvaccinated control group. Following viral challenge, two vaccinated animals, one receiving DNA alone and one the prime-boost vaccine remained free of viraemia, whilst all controls became viraemic. Animals vaccinated with WIV showed apparent early enhancement of infection at 2 weeks post challenge (pc) with higher plasma viral RNA loads than control animals or cats immunised with DNA alone. Despite this, animals vaccinated with WIV or DNA alone showed significantly lower proviral loads in peripheral blood mononuclear cells and mesenteric lymph node cells, whilst those receiving the DNA-WIV prime-boost vaccine showed significantly lower proviral loads in PBMC, than control animals, at 35 weeks pc. Therefore both DNA and WIV vaccines conferred limited protection against viral challenge but the combination of WIV and DNA in a prime-boost approach appeared to offer no significant advantage over either vaccine alone.

Lessons from the cat: development of vaccines against lentiviruses.

Feline immunodeficiency virus (FIV) is a natural infection of domestic cats, which produces a disease with many similarities to human immunodeficiency virus (HIV) infection in man. The virus is an important cause of morbidity and mortality in pet cats worldwide. As such an effective vaccine is desirable both for its use in veterinary medicine and also as a model for the development of an HIV vaccine. A large number of candidate vaccines have been tested against feline immunodeficiency virus. These include inactivated virus and infected cell vaccines, DNA and viral vectored vaccines, subunit and peptide vaccines and vaccines using bacterial vectors. Ultimately, the development of inactivated virus and infected cell vaccines led to the release of the first licensed vaccine against FIV, in 2002. This review highlights some of the difficulties associated with the development of lentiviral vaccines and some of the lessons that have been learned in the FIV model that are of particular relevance to the development of HIV vaccines.

Isolation, expression and bioactivity of feline granulocyte-macrophage colony-stimulating factor.

A cDNA encoding feline granulocyte-macrophage colony stimulating factor was cloned from alveolar macrophages using the reverse transcriptase-polymerase chain reaction (RT-PCR). The cDNA is 426 bp in length and encodes a predicted mature protein of 127 amino acids and the majority of the signal peptide. The recombinant protein (rfGM-CSF) was expressed in both Escherichia coli, as a calmodulin fusion protein, and mammalian cells. Biological activity of both recombinant proteins was demonstrated using the human erythroleukaemic cell line, TF-1. In a soft agar clonogenic assay, rfGM-CSF supported the development of granulocyte, macrophage and granulocyte-macrophage colonies. In combination with phytohaemagglutin (PHA) lymphocyte-conditioned medium, the number and size of such colonies were increased. Culture of feline bone marrow cells with rfGM-CSF was an efficient method for producing cells with morphology typical of dendritic cells (DC). The availability of the recombinant cytokine will permit further studies, in particular, the evaluation of the role of dendritic cells in feline immunopathology and its potential as a vaccine adjuvant.

The application of nucleic acid vaccines in veterinary medicine.

Nucleic acid immunisation entails the delivery of DNA (or RNA) encoding a vaccine antigen to the recipient. The DNA is taken up by host cells and transcribed to mRNA, from which the vaccine proteins are then translated. The expressed proteins are recognised as foreign by the host immune system and elicit an immune response, which may have both cell-mediated and humoral components. DNA vaccines offer a number of advantages over conventional vaccines, including ease of production, stability and cost. They also allow the production of vaccines against organisms which are difficult or dangerous to culture in the laboratory. This review describes the principles of DNA vaccination and the application of DNA vaccines to veterinary species. Although a great deal of developmental work is required before the technology can give rise to commercial vaccines in domestic animals, there is ongoing research in many fields and it is expected that a number of exciting developments will arise in the next decade.

Longitudinal analysis of feline leukemia virus-specific cytotoxic T lymphocytes: correlation with recovery from infection.

Feline leukemia virus (FeLV) is a common naturally occurring gammaretrovirus of domestic cats that is associated with degenerative diseases of the hematopoietic system, immunodeficiency, and neoplasia. Although the majority of cats exposed to FeLV develop a transient infection and recover, a proportion of cats become persistently viremic and many subsequently develop fatal diseases. To define the dominant host immune effector mechanisms responsible for the outcome of infection, we studied the longitudinal changes in FeLV-specific cytotoxic T lymphocytes (CTLs) in a group of naïve cats following oronasal exposure to FeLV. Using (51)Cr release assays to measure ex vivo virus-specific cytotoxicity, the emerging virus-specific CTL response was correlated with modulations in viral burden as assessed by detection of infectious virus, FeLV p27 capsid antigen, and proviral DNA in the blood. High levels of circulating FeLV-specific effector CTLs appeared before virus neutralizing antibodies in cats that recovered from exposure to FeLV. In contrast, persistent viremia was associated with a silencing of virus-specific humoral and cell-mediated host immune effector mechanisms. A single transfer of between 2 x 10(7) and 1 x 10(8) autologous, antigen-activated lymphoblasts was associated with a downmodulation in viral burden in vivo. The results suggest an important role for FeLV-specific CTLs in retroviral immunity and demonstrate the potential to modulate disease outcome by the adoptive transfer of antigen-specific T cells in vivo.

Protection against feline immunodeficiency virus using replication defective proviral DNA vaccines with feline interleukin-12 and -18.

A molecular clone of the Glasgow-8 isolate of FIV (FIVGL8) was rendered replication defective by an in-frame deletion in either reverse transcriptase (deltaRT) or integrase (deltaIN) genes for use as DNA vaccines. To test the ability of these multi-gene vaccines to protect against two feline immunodeficiency virus (FIV) isolates of differing virulence, cats were immunized using either DNA vaccine alone or co-administered with interleukin-12 (IL-12) and/or interleukin-18 (IL-18) cytokine DNA. Animals were challenged sequentially with FIV-Petaluma (FIVPET) an FIV isolate of relatively low virulence and subsequently with the more virulent FIVGL8. A proportion of vaccinates (5/18 deltaIN and 2/12 deltaRT) were protected against primary challenge with FIV(PET). Five of the vaccinated-protected cats were re-challenged with FIV(PET); four (all deltaIN) remained free of viraemia whilst all naive controls became viraemic. Following subsequent challenge with the more virulent FIVGL8 these four vaccinated-protected animals all became viraemic but showed lower proviral loads than naive cats. This study suggests that while our current DNA vaccines may not produce sterilizing immunity against more virulent isolates of FIV, they may nevertheless significantly reduce the impact of infection.