Reverse mutation of the virulence-associated S2 gene does not cause an attenuated equine infectious anemia virus strain to revert to pathogenicity.

The contribution of S2 accessory gene of equine infectious anemia virus (EIAV) to the virulence of pathogenic strains was investigated in the present study by reverse mutation of all four consensus S2 mutation sites in an attenuated EIAV proviral strain, FDDV3-8, to the corresponding sequences of a highly pathogenic strain DV117. The S2 reverse-mutated recombinant strain FDDVS2r1-2-3-4 replicated with similar kinetics to FDDV3-8 in cultivated target cells. In contrast to the results of other studies of EIAV with dysfunctional S2, reverse mutation of S2 only transiently and moderately increased the plasma viral load of inoculated horses, and induction of transient immunosuppression did not boost viral pathogenicity. In addition, inoculation of FDDVS2r1-2-3-4 induced partial protection to a challenge pathogenic virus. These results suggest that the attenuated EIAV vaccine strain with multiple mutations in multiple genes will not easily revert to a virulent phenotype.

A pilot study comparing the development of EIAV Env-specific antibodies induced by DNA/recombinant vaccinia-vectored vaccines and an attenuated Chinese EIAV vaccine.

Data from successful attenuated lentiviral vaccine studies indicate that fully mature Env-specific antibodies characterized by high titer, high avidity, and the predominant recognition of conformational epitopes are associated with protective efficacy. Although vaccination with a DNA prime/recombinant vaccinia-vectored vaccine boost strategy has been found to be effective in some trials with non-human primate/simian/human immunodeficiency virus (SHIV) models, it remains unclear whether this vaccination strategy could elicit mature equine infectious anemia virus (EIAV) Env-specific antibodies, thus protecting vaccinated horses against EIAV infection. Therefore, in this pilot study we vaccinated horses using a strategy based on DNA prime/recombinant Tiantan vaccinia (rTTV)-vectored vaccines encoding EIAV env and gag genes, and observed the development of Env-specific antibodies, neutralizing antibodies, and p26-specific antibodies. Vaccination with DNA induced low titer, low avidity, and the predominant recognition of linear epitopes by Env-specific antibodies, which was enhanced by boosting vaccinations with rTTV vaccines. However, the maturation levels of Env-specific antibodies induced by the DNA/rTTV vaccines were significantly lower than those induced by the attenuated vaccine EIAV(FDDV). Additionally, DNA/rTTV vaccines did not elicit broadly neutralizing antibodies. After challenge with a virulent EIAV strain, all of the vaccinees and control horses died from EIAV disease. These data indicate that the regimen of DNA prime/rTTV vaccine boost did not induce mature Env-specific antibodies, which might have contributed to immune protection failure.

An attenuated EIAV vaccine strain induces significantly different immune responses from its pathogenic parental strain although with similar in vivo replication pattern.

The EIAV (equine infectious anemia virus) multi-species attenuated vaccine EIAV(DLV121) successfully prevented the spread of equine infectious anemia (EIA) in China in the 1970s and provided an excellent model for the study of protective immunity to lentiviruses. In this study, we compared immune responses induced by EIAV(DLV121) to immunity elicited by the virulent EIAV(LN40) strain and correlated immune responses to protection from infection. Horses were randomly grouped and inoculated with either EIAV(DLV121) (Vaccinees, Vac) or a sublethal dose of EIAV(LN40) (asymptomatic carriers, Car). Car horses became EIAV(LN40) carriers without disease symptoms. Two of the four Vac horses were protected against infection and the other two had delayed onset or reduced severity of EIA with a lethal EIAV(LN40) challenge 5.5 months post initial inoculation. In contrast, all three Car animals developed acute EIA and two succumbed to death. Specific humoral and cellular immune responses in both Vac and Car groups were evaluated for potential correlations with protection. These analyses revealed that although plasma viral loads remained between 10(3) and 10(5)copies/ml for both groups before EIAV(LN40) challenge, Vac-treated animals developed significantly higher levels of conformational dependent, Env-specific antibody, neutralizing antibody as well as significantly elevated CD4(+) T cell proliferation and IFN-γ-secreting CD8(+) T cells than those observed in EIAV(LN40) asymptomatic carriers. Further analysis of protected and unprotected cases in vaccinated horses identified that cellular response parameters and the reciprocal anti-p26-specific antibody titers closely correlated with protection against infection with the pathogenic EIAV(LN40). These data provide a better understanding of protective immunity to lentiviruses.

A pilot study on an attenuated Chinese EIAV vaccine inducing broadly neutralizing antibodies.

The attenuated Chinese equine infectious anemia virus (EIAV) vaccine has successfully protected millions of equine animals from EIA disease in China. In this pilot study, to determine whether this attenuated vaccine can induce broadly neutralizing antibodies, we immunized four horses with the attenuated Chinese vaccine strain EIAVFDDV and then observed the evolution of neutralizing antibodies against different EIAV strains. During the vaccination phase, all vaccinees rapidly developed high levels of neutralizing antibodies against the homologous vaccine strain (pLGFD3V), and 3 out of 4 horses showed a gradual increase in serum neutralizing activity against two relatively heterologous virulent variants of the challenge strain (pLGFD3Mu12V and DLV34). After challenge, the three horses that had developed high levels of neutralizing antibodies against pLGFD3Mu12V and DLV34 did not show signs of infection, which was demonstrated by immune suppression, while the one horse producing serum that could only neutralize pLGFD3V developed a febrile episode during the 8-month observation period. To assess whether the broadly neutralizing activity is associated with immune protection, sera drawn on the day of challenge from these four vaccinees and an additional four EIAVFDDV-vaccinated horses were analyzed for neutralizing antibodies against pLGFD3V, pLGFD3Mu12V and DLV34. Although there was no significant correlation between protection from infection and serum neutralizing activity against any of these three viral strains, protection from infection was observed to correlate better with serum neutralizing activity against the two heterologous virulent strains than against the homologous vaccine strain. These data indicate that EIAVFDDV induced broadly neutralizing antibodies, which might confer enhanced protection of vaccinees from infection by the challenge virus.

Unique evolution characteristics of the envelope protein of EIAV(LN40), a virulent strain of equine infectious anemia virus.

The Chinese equine infectious anemia virus (EIAV) virulent strain EIAV(LN40) is derived from a naturally occurring virus by continuously passing in horses for 16 generations. Its genome sequence is 23% different from that of the American strains or the Japanese strains, and the variation of envelope gp90 surface unit (SU) is as high as 41%. In this study, evolutions of the EIAV(LN40) gp90 gene in four infected horses were analyzed. Results showed that new quasispecies arose in the early stage of infection in all EIAV(LN40)-infected horses. These quasispecies belonged to branches different from EIAV(LN40) in a phylogenetic tree. In contrast, the gp90 sequences of viruses isolated after disease onset remained in the same phylogenetic branch as EIAV(LN40), with some having exactly the same sequences. The glycosylation sites 191NSSN and 237NNTW in the V3 and V4 region present or absent simultaneously in most of the predicted amino acid sequences. Changes in the glycosylation sites within V3, V4, and V5 regions are usually associated with the disease status. Glycosylation sites (191NSSN, 237NNTW, and 280NDTS) within these three regions were present in EIAV(LN40) and most of the quasispecies isolated after, but not before disease onset. These unique evolutionary characteristics of SU have not been reported for EIAV and other lentiviruses. Our results provide a reference for a further understanding of the mechanism underlying the persistent infection and escape from immune surveillance of EIAV.

A proviral derivative from a reference attenuated EIAV vaccine strain failed to elicit protective immunity.

To investigate essential factors that determine the efficacy of vaccines against lentiviruses, an effective attenuated equine infectious anemia virus (EIAV) vaccine strain and a proviral derivative of the vaccine were compared with respect to differences in inducing protective immunity. Although these two strains replicated equally well in vitro and in vivo, the proviral strain induced significantly less protection from disease and infection caused by viral challenge and significantly lower specific neutralizing capability. These findings indicated that the proviral strain had lost the ability to stimulate immune protection compared to the parental vaccine strain. A further analysis of the envelope gp90 gene variation revealed that compared to the proviral strain, the vaccine strain displayed a wide sequence diversity in immunogen composition. Thus, we inferred that the differences in immunogen composition might be the major cause for the failure of the proviral derivative to elicit the immune protection induced by the parental strain.

Genomic comparison between attenuated Chinese equine infectious anemia virus vaccine strains and their parental virulent strains.

A lentiviral vaccine, live attenuated equine infectious anemia virus (EIAV) vaccine, was developed in the 1970s, and this has made tremendous contributions to the control of equine infectious anemia (EIA) in China. Four key virus strains were generated during the attenuation of the EIAV vaccine: the original Liao-Ning strain (EIAV(LN40)), a donkey-adapted virulent strain (EIAV(DV117)), a donkey-leukocyte-attenuated vaccine strain (EIAV(DLV121)), and a fetal donkey dermal cell (FDD)-adapted vaccine strain (EIAV(FDDV13)). In this study, we analyzed the proviral genomes of these four EIAV strains and found a series of consensus substitutions among these strains. These mutations provide useful information for understanding the genetic basis of EIAV attenuation. Our results suggest that multiple mutations in a variety of genes in our attenuated EIAV vaccines not only provide a basis for virulence attenuation and induction of protective immunity but also greatly reduce the risk of reversion to virulence.

Amino acid mutations in the env gp90 protein that modify N-linked glycosylation of the Chinese EIAV vaccine strain enhance resistance to neutralizing antibodies.

The Chinese EIAV vaccine is an attenuated live-virus vaccine obtained by serial passage of a virulent horse isolate (EIAV(L)) in donkeys (EIAV(D)), and subsequently in donkey cells in vitro. In this study, we compare the env gene of the original horse virulent virus (EIAV(L)) with attenuated strains serially passaged in donkey MDM (EIAV(DLV)), and donkey dermal cells (EIAV(FDDV)). Genetic comparisons among parental and attenuated strains found that vaccine strains contained amino acid substitutions/deletions in gp90 that resulted in a loss of three potential N-linked glycosylation sites, designated g5, g9, and g10. To investigate the biological significance of these changes, reverse-mutated viruses were constructed in the backbone of the EIAV(FDDV) infectious molecular clone (pLGFD3). The resulting virus stocks were characterized for replication efficiency in donkey dermal cells and donkey MDM, and were tested for sensitivity to neutralization using sera from two ponies experimentally infected with EIAV(FDDV). The results clearly show that these mutations generated by site-directed mutagenesis resulted in cloned viruses with enhanced resistance to serum-neutralizing antibodies that were also able to recognize parental viruses. The results of this study indicate that these mutations play an important role in the attenuation of the EIAV vaccine strains.

Genomic analysis of an effective lentiviral vaccine-attenuated equine infectious anemia virus vaccine EIAV FDDV13.

Chinese equine infectious anemia virus (EIAV) attenuated vaccine is the first lentiviral vaccine with a successful application. In order to understand the correlation of viral genomic mutations with viral attenuation and with induced immunoprotective properties, we analyzed the proviral genome sequences of the EIAV-attenuated vaccine strain EIAV(FDDV13) (EIAV fetal donkey dermal cell-adapted vaccine) and its highly virulent parental strain EIAV(LN40). The sequences of these strains were compared with those of the major foreign EIAV strains. The results indicated a large genetic distance between the Chinese EIAV strain and the major EIAV strains in America and Japan. The Chinese strains belong to an independent phylogenetic branch. The divergence between the entire genome of the Chinese strains and that of other major EIAV strains is approximately 23%. The divergence rate in LTR is over 14%, whereas that in each open reading frame is over 20%. The gp90 exhibited a divergence of 35% in its nucleotide sequence and 40% in its amino acid sequence. The present study found that after long-term passage in vitro, EIAV(FDDV13) has accumulated many stable substitution mutations in each gene. These mutations at multiple sites in multiple genes of the vaccine strain, especially the conserved mutations, provide important references for further understanding the attenuation mechanism of Chinese EIAV-attenuated vaccine and the immunoprotection mechanism of lentiviral vaccines.

[Construction and in vitro evaluation of an infectious clone of the equine infectious anemia virus vaccine strain EIAV(FDDV) with four reverse-mutated vaccine-specific sites in the S2 gene].

To elucidate the function of the S2 gene in equine infectious anemia virus (EIAV) and its role in the attenuation of the Chinese attenuated EIAV vaccine strains, the S2 in the EIAV vaccine strain EIAV (FDDV) was reverse-mutated and the in vitro replication character of the resultant virus was evaluated. Based on the sequence variation of the S2 gene between the EIAV virulent strains and vaccine strains, all the four vaccine-specific sites in the S2 of an EIAV(FDDV) infectious clone, pFDDV3-8, were reverse-mutated to the sequences of the virulent strain EIAV(DV115). The reverse-mutated molecular clone pFDDVS2r1-3-4-5 was used to transfect fetal donkey dermal (FDD) cells for rescuing the derived virus vpFDDVS2r1-3-4-5. The production and replication of vpFDDVS2r1-3-4-5 in FDD cells were proved by RT-PCR, immune fluorescence assay and reverse transcriptase activity assay. Typical virons of EIAV were clearly observed under the electron microscopy. The parallel analysis of the dynamic replication of the reverse-mutated viral clone vpFDDVS2r1-3-4-5 and its parental virus vpFDDV3-8 showed that the virus with four reverse mutations in the S2 replicated only slightly slower than its parental vaccine strain in FDD cells. This result implicates that the mutations in the S2 of the EIAV vaccine strains did not significantly alter the viral replication in vitro. Further studies on the in vivo replication of the reverse-mutated viral clone are required for understanding the relationship between the S2 and the attenuated pathogenesis of EIAV attenuated vaccines.

In vivo evolution of the gp90 gene and consistently low plasma viral load during transient immune suppression demonstrate the safety of an attenuated equine infectious anemia virus (EIAV) vaccine.

To study the in vivo evolution of the attenuated Chinese equine infectious anemia virus (EIAV) vaccine, viral gp90 gene variation and virus replication in immunosuppressed hosts were investigated. The results showed that after vaccination, the gp90 gene followed an evolutionary trend of declining diversity. The trend coincided with the maturation of immunity to EIAV, and eventually, the gp90 gene became highly homologous. The sequences of these predominant quasispecies were consistently detected up to 18 months after vaccination. Furthermore, after transient immune suppression with dexamethasone, the plasma viral RNA copy number of the vaccine strain in three vaccinated ponies remained consistently below the "pathogenic threshold" level, while the viral load increased by 25,000-fold in the positive control of an inapparent carrier of the parental virulent strain. This study is the first to provide evidence for the safety of an attenuated lentiviral vaccine with decreased genomic diversity and consistently low viral replication under suppressed immunity.

Genetic variation in the long terminal repeat associated with the transition of Chinese equine infectious anemia virus from virulence to avirulence.

A highly virulent strain of equine infectious anemia virus (EIAV) lost its fatal virulence but retained the desired antigens during serial passage over 130 generations in leukocytes in vitro. We compared the long terminal repeat (LTR) sequences of the different generations and found that three stable genetic variations occurred in the transcriptional start site, the initial base of TAR, and the pre-mRNA cleavage site at the R-U5 boundary, respectively. These three mutations happened at the inflexion of virus pathogenicity loss; therefore, the function of these mutations needs to be further addressed.

Effective inhibition of infectious bursal disease virus replication in vitro by DNA vector-based RNA interference.

Infectious bursal disease (IBD) leads to considerable economic losses for the poultry industry by inducing severe immunosuppression and high mortality in chickens. The objective of this study was to determine if RNA interference (RNAi) could be utilized to inhibit IBDV replication in vitro. We selected 3 short interfering RNA (siRNA) sequences (siVP1(618), siVP1(1,115), and siVP1(2,571)) based on conserved regions in the vp1 gene of the infectious bursal disease virus (IBDV). When the Vero cells were transfected with siRNA, synthesized via in vitro transcription, and then infected with IBDV, siVP1(2,571) was discovered to be the most effective site for inhibiting IBDV replication. For long-term expression of siRNA and due to its suitability for large-scale preparation, the mouse U6 promoter was amplified using primers designed according to the siVP1(2,571) sequence. The resulting products were then subcloned into pEGFP-C1 to construct the shRNA expression vector pEC2571-shRNA. The shRNA-transfected Vero cells were then infected with IBDV. As compared to the control, the inhibitory rate in the pEC2,571-shRNA-transfected group was 87.4%. Indirect immunofluorescence and real-time polymerase chain reaction (PCR) confirmed that VP1 expression decreased at both the protein and RNA levels as compared to that in the controls. The results presented here indicate that DNA vector-based RNAi could effectively inhibit IBDV replication in vitro.

Correlation between the induction of Th1 cytokines by an attenuated equine infectious anemia virus vaccine and protection against disease progression.

The equine infectious anemia virus (EIAV) donkey-leukocyte attenuated vaccine (DLV) has been used to protect against equine infectious anaemia (EIA) disease for several decades in China. The attenuated mechanism and immunological protective mechanisms remain to be elucidated. To identify responses that correlate with the protection against disease, we immunized horses with DLV, followed by challenge with an EIAV wild-type strain LN. All vaccinated horses were asymptomatic and had a low level of virus replication (<10 copies ml-1). The expression level of cytokines including gamma interferon, interleukin 2 and 12 in DLV immunized horses was 5-100-fold higher than that in non-vaccinated controls (n=4, P<0.01). After challenge with virulent LN, horses vaccinated with DLV showed lower viral loads (<10(3) copies ml-1) with no temperature increase, except for one transient febrile episode in one animal. In contrast, horses in the non-vaccinated control group experienced much higher viral loads (>10(7) copies ml-1) and intermittent febrile episodes. Cytokine production in the DLV-vaccinated horses increased and attained a plateau level at approximately 50 days post-vaccination, and exceeded 10(7) copies per 10(7) peripheral blood mononuclear cells (PBMCs) 1-3 months post-challenge. However, non-vaccinated control horses died after several fever episodes (>or=39 degrees C), which coincided with higher viral load (10(6)-10(7) copies ml-1) and lower cytokine production (<10(4) copies per 10(7) PBMCs). The results indicate that high levels of EIAV-specific cytokines induced by the attenuated EIAV vaccine may contribute to the protective immune response against EIA disease.

[Elevation of IFN-gamma transcription level in peripheral blood mononuclear cells of EIAV vaccinated horses].

AIM: To evaluate the relationship between the transcriptional level of IFN-gamma mRNA in peripheral blood mononuclear cells (PBMC) and immune protective response driven by inoculated horses with donkey leukocyte attenuated vaccine of EIAV(DLV), and to elucidate the immune mechanism of DLV. METHODS: A real-time PCR method was established for quantitative detection of IFN-gamma mRNA level from horse PBMCs. Twelve horses were divided into vaccination group, healthy control group, challenging control group and EIAV natural infection group. The transcriptional level and distribution of IFN-gamma mRNA in PBMCs were analyzed. The temperature and other parameters of the inoculated horses were monitored or a daily basis. Horses inoculated with DLV vaccines were challenged post-inoculated 8 months, and the change of transcriptional level of IFN-gamma mRNA during pre/post-challenge was evaluated. RESULTS: Compared with the healthy control and natural infection group. The transcriptional level of IFN-gamma mRNA was much higher in vaccination group (P<0.01), and kept increasing even after challenging with virulent EIAV strains. In the challenging control group, the transcriptional level of IFN-gamma mRNA fluctuated with the development of disease and reduced markedly during febrific episodes. CONCLUSION: This study for the first time reveals that EIAV-attenuated vaccine could induce high level transcription of IFN-gamma mRNA, with is associated closely with immune protective response induced by DLV. This finding provides some clues to elucidate the mechanism of immunity protection of DLV at the molecular level.

[Induction of EIAV-specific cellular immune response by attenuated EIAV vaccine].

AIM: To elucidate cellular immune protective mechanism of EIAV. METHODS: Four horses were immunized with (DLV) by subcutaneous injection and 2 horses with 0.85% sodium chloride as the negative control. Rectal temperatures and clinical features were recorded daily. Whole blood samples were collected, from which PBMC were separated and used for CTL assay and lymphocyte proliferation assay. RESULTS: The target cells were activated by PWM and treated with DLV or recombinant vaccinia vectors expressing DLV and LN Gag or Env separately. The percentage of EIAV-specific CTL lysis was under 5% in the negative control group, however, that was 20% to 30% in the group of immunized horses. The stimulation index (SI) from proliferation assay was 3 in the group immunized with attenuated DLV vaccine. CONCLUSION: The results indicated that EIAV-specific cellular immune-response induced by DLV immunization may contribute to the immune protective effect against EIAV virus infection.

Amino acid mutations of the infectious clone from Chinese EIAV attenuated vaccine resulted in reversion of virulence.

The Chinese equine infectious anemia virus (EIAV) donkey-leukocyte attenuated vaccine (DLV) provides a unique natural model system by which attenuated mechanism and immunological control of lentivirus replication may be studied. We analyzed the critical consensus mutations that occurred during the viral passages in vitro and in vivo for vaccine's preparation. Based on the full-length infectious clone pLGFD3 (EIAV vaccine background) and according to mutations displayed during viral attenuation, we successfully constructed an infectious clones pLG5-3-l in which gag and env genes were point-mutated by overlap PCR mutagenesis strategy. pLG5-3-l was proved to have the ability of effective replication in vitro cells culture systems by Reverse Transcriptase Assay and virion observation under electron microscopy. Results of the in vivo experiments indicated that marked differences occurred between the mutated virus and their parental virus in clinical manifestation and plasma viral replication during 6-month observation period. In contrast to asymptom of animals infected with pLGFD3-V, the mutated virus (pLG5-3-l-V) developed typical clinical progression in the corresponding experimentally infected animals. The results of the distinct differences in clinical profiles and viral dynamics before and after mutation of EIAV infectious clone will help to understand the protective mechanism of Chinese EIAV vaccine and shed light on novel HIV vaccine design.