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Effective T cell induction is an important strategy in HIV-vaccine development. However, it has been indicated that vaccine-induced HIV-specific CD4

Effective vaccines are essential for the control of the COVID-19 pandemic. Currently-developed vaccines inducing SARS-CoV-2 spike (S) antigen-specific neutralizing antibodies (NAbs) are effective, but the appearance of NAb-resistant S variant viruses is of great concern. A vaccine inducing S-independent or NAb-independent SARS-CoV-2 control may contribute to containment of these variants. Here, we investigate the efficacy of an intranasal vaccine expressing viral non-S antigens against intranasal SARS-CoV-2 challenge in cynomolgus macaques. Seven vaccinated macaques exhibit significantly reduced viral load in nasopharyngeal swabs on day 2 post-challenge compared to nine unvaccinated controls. The viral control in the absence of SARS-CoV-2-specific NAbs is significantly correlated with vaccine-induced viral antigen-specific CD8+ T-cell responses. Our results indicate that CD8+ T-cell induction by intranasal vaccination can result in NAb-independent control of SARS-CoV-2 infection, highlighting a potential of vaccine-induced CD8+ T-cell responses to contribute to COVID-19 containment., Graphical Abstract, Ishii et al. show neutralization-independent SARS-CoV-2 control associated with vaccine-induced CD8+ T-cell responses, indicating that virus-specific CD8+ T-cell induction by intranasal vaccination can result in SARS-CoV-2 control. Results highlight the potential of vaccine-induced CD8+ T-cell responses to contribute to the control of SARS-CoV-2 variants.

SARS-CoV-2 infection presents clinical manifestations ranging from asymptomatic to fatal respiratory failure. Despite the induction of functional SARS-CoV-2-specific CD8+ T-cell responses in convalescent individuals, the role of virus-specific CD8+ T-cell responses in the control of SARS-CoV-2 replication remains unknown. In the present study, we show that subacute SARS-CoV-2 replication can be controlled in the absence of CD8+ T cells in cynomolgus macaques. Eight macaques were intranasally inoculated with 105 or 106 TCID50 of SARS-CoV-2, and three of the eight macaques were treated with a monoclonal anti-CD8 antibody on days 5 and 7 post-infection. In these three macaques, CD8+ T cells were undetectable on day 7 and thereafter, while virus-specific CD8+ T-cell responses were induced in the remaining five untreated animals. Viral RNA was detected in nasopharyngeal swabs for 10-17 days post-infection in all macaques, and the kinetics of viral RNA levels in pharyngeal swabs and plasma neutralizing antibody titers were comparable between the anti-CD8 antibody treated and untreated animals. SARS-CoV-2 RNA was detected in the pharyngeal mucosa and/or retropharyngeal lymph node obtained at necropsy on day 21 in two of the untreated group but undetectable in all macaques treated with anti-CD8 antibody. CD8+ T-cell responses may contribute to viral control in SARS-CoV-2 infection, but our results indicate possible containment of subacute viral replication in the absence of CD8+ T cells, implying that CD8+ T-cell dysfunction may not solely lead to viral control failure.Author SummarySARS-CoV-2 infection presents a wide spectrum of clinical manifestations ranging from asymptomatic to fatal respiratory failure. The determinants for failure in viral control and/or fatal disease progression have not been elucidated fully. Both acquired immune effectors, antibodies and CD8+ T cells, are considered to contribute to viral control. However, it remains unknown whether a deficiency in either of these two arms is directly linked to failure in the control of SARS-CoV-2 replication. In the present study, to know the requirement of CD8+ T cells for viral control after the establishment of infection, we examined the effect of CD8+ cell depletion by monoclonal anti-CD8 antibody administration in the subacute phase on SARS-CoV-2 replication in cynomolgus macaques. Unexpectedly, our analysis revealed no significant impact of CD8+ cell depletion on viral replication, indicating that subacute SARS-CoV-2 replication can be controlled in the absence of CD8+ T cells. CD8+ T-cell responses may contribute to viral control in SARS-CoV-2 infection, but this study suggests that CD8+ T-cell dysfunction may not solely lead to viral control failure or fatal disease progression.

SARS-CoV-2 infection presents clinical manifestations ranging from asymptomatic to fatal respiratory failure. Despite the induction of functional SARS-CoV-2-specific CD8+ T-cell responses in convalescent individuals, the role of virus-specific CD8+ T-cell responses in the control of SARS-CoV-2 replication remains unknown. In the present study, we show that subacute SARS-CoV-2 replication can be controlled in the absence of CD8+ T cells in cynomolgus macaques. Eight macaques were intranasally inoculated with 105 or 106 TCID50 of SARS-CoV-2, and three of the eight macaques were treated with a monoclonal anti-CD8 antibody on days 5 and 7 post-infection. In these three macaques, CD8+ T cells were undetectable on day 7 and thereafter, while virus-specific CD8+ T-cell responses were induced in the remaining five untreated animals. Viral RNA was detected in nasopharyngeal swabs for 10–17 days post-infection in all macaques, and the kinetics of viral RNA levels in pharyngeal swabs and plasma neutralizing antibody titers were comparable between the anti-CD8 antibody treated and untreated animals. SARS-CoV-2 RNA was detected in the pharyngeal mucosa and/or retropharyngeal lymph node obtained at necropsy on day 21 in two of the untreated group but undetectable in all macaques treated with anti-CD8 antibody. CD8+ T-cell responses may contribute to viral control in SARS-CoV-2 infection, but our results indicate possible containment of subacute viral replication in the absence of CD8+ T cells, implying that CD8+ T-cell dysfunction may not solely lead to viral control failure., Author summary SARS-CoV-2 infection presents a wide spectrum of clinical manifestations ranging from asymptomatic to fatal respiratory failure. The determinants for failure in viral control and/or fatal disease progression have not been elucidated fully. Both acquired immune effectors, antibodies and CD8+ T cells, are considered to contribute to viral control. However, it remains unknown whether a deficiency in either of these two arms is directly linked to failure in the control of SARS-CoV-2 replication. In the present study, to know the requirement of CD8+ T cells for viral control after the establishment of infection, we examined the effect of CD8+ cell depletion by monoclonal anti-CD8 antibody administration in the subacute phase on SARS-CoV-2 replication in cynomolgus macaques. Unexpectedly, our analysis revealed no significant impact of CD8+ cell depletion on viral replication, indicating that subacute SARS-CoV-2 replication can be controlled in the absence of CD8+ T cells. CD8+ T-cell responses may contribute to viral control in SARS-CoV-2 infection, but this study suggests that CD8+ T-cell dysfunction may not solely lead to viral control failure or fatal disease progression.

Anti-retroviral therapy (ART) can inhibit HIV proliferation but not achieve virus eradication from HIV-infected individuals. Under ART-based HIV control, virus-specific CD8+ T-cell responses are often reduced. Here, we investigated the impact of therapeutic vaccination inducing virus-specific CD8+ T-cell responses under ART on viral control in a macaque AIDS model. Twelve rhesus macaques received ART from week 12 to 32 after simian immunodeficiency virus (SIV) infection. Six of them were vaccinated with Sendai virus vectors expressing SIV Gag and Vif at weeks 26 and 32, and Gag/Vif-specific CD8+ T-cell responses were enhanced and became predominant. All macaques controlled viremia during ART but showed viremia rebound after ART cessation. Analysis of in vitro CD8+ cell ability to suppress replication of autologous lymphocytes-derived SIVs found augmentation of anti-SIV efficacy of CD8+ cells after vaccination. In the vaccinated animals, the anti-SIV efficacy of CD8+ cells at week 34 was correlated positively with Gag-specific CD8+ T-cell frequencies and inversely with rebound viral loads at week 34. These results indicate that Gag-specific CD8+ T-cell induction by therapeutic vaccination can augment anti-virus efficacy of CD8+ cells, which may be insufficient for functional cure but contribute to more stable viral control under ART.

Optimization of immunogen is crucial for induction of effective T-cell responses in the development of a human immunodeficiency virus (HIV) vaccine. Conventional T-cell-based vaccines have been designed to induce virus-specific CD4+ T as well as CD8+ T cells. However, it has been indicated that induction of HIV-specific CD4+ T cells, preferential targets for HIV infection, by vaccination may be detrimental and accelerate viral replication after HIV exposure. In the present study, we present a novel immunogen to selectively induce CD8+ T cells but not CD4+ T cells targeting viral antigens. The immunogen, CaV11, was constructed by tandem connection of overlapping 11-mer peptides spanning simian immunodeficiency virus (SIV) Gag capsid (CA) and Vif. Prime-boost immunization with DNA and Sendai virus (SeV) vectors expressing CaV11 efficiently induced Gag/Vif-specific CD8+ T-cell responses with inefficient Gag/Vif-specific CD4+ T-cell induction in rhesus macaques (n = 6). None of the macaques exhibited the enhancement of acute viral replication after an intravenous high-dose SIV challenge, which was observed in those immunized with DNA and SeV expressing the whole Gag protein in our previous study. Set point viral control postinfection was associated with SeV-specific CD4+ T-cell responses postimmunization, suggesting contribution of SeV-specific helper responses to effective Gag/Vif-specific CD8+ T-cell induction by vaccination. This immunogen design could be a promising method for selective induction of effective anti-HIV CD8+ T-cell responses.IMPORTANCE Induction of effective CD8+ T-cell responses is an important HIV vaccine strategy. Several promising vaccine delivery tools have been developed, and immunogen optimization is now crucial for effective T-cell induction. Conventional immunogens have been designed to induce virus-specific CD4+ T cells as well as CD8+ T cells, but induction of virus-specific CD4+ T cells that are preferential targets for HIV infection could enhance acute HIV proliferation. Here, we designed a novel immunogen to induce HIV-specific CD8+ T cells without HIV-specific CD4+ T-cell induction but with non-HIV antigen-specific CD4+ T-cell help. Our analysis in a macaque AIDS model showed that our immunogen can efficiently elicit effective CD8+ T but not CD4+ T cells targeting viral antigens, resulting in no enhancement of acute viral replication after virus exposure. This immunogen design, also applicable for other currently developed immunogens, could be a promising method for selective induction of effective anti-HIV CD8+ T-cell responses.

Anti-retroviral therapy (ART) can inhibit HIV proliferation but not achieve virus eradication from HIV-infected individuals. Under ART-based HIV control, virus-specific CD8+ T-cell responses are often reduced. Here, we investigated the impact of therapeutic vaccination inducing virus-specific CD8+ T-cell responses under ART on viral control in a macaque AIDS model. Twelve rhesus macaques received ART from week 12 to 32 after simian immunodeficiency virus (SIV) infection. Six of them were vaccinated with Sendai virus vectors expressing SIV Gag and Vif at weeks 26 and 32, and Gag/Vif-specific CD8+ T-cell responses were enhanced and became predominant. All macaques controlled viremia during ART but showed viremia rebound after ART cessation. Analysis of in vitro CD8+ cell ability to suppress replication of autologous lymphocytes-derived SIVs found augmentation of anti-SIV efficacy of CD8+ cells after vaccination. In the vaccinated animals, the anti-SIV efficacy of CD8+ cells at week 34 was correlated positively with Gag-specific CD8+ T-cell frequencies and inversely with rebound viral loads at week 34. These results indicate that Gag-specific CD8+ T-cell induction by therapeutic vaccination can augment anti-virus efficacy of CD8+ cells, which may be insufficient for functional cure but contribute to more stable viral control under ART.

Optimization of immunogen is crucial for induction of effective T-cell responses in the development of a human immunodeficiency virus (HIV) vaccine. Conventional T-cell-based vaccines have been designed to induce virus-specific CD4(+) T as well as CD8(+) T cells. However, it has been indicated that induction of HIV-specific CD4(+) T cells, preferential targets for HIV infection, by vaccination may be detrimental and accelerate viral replication after HIV exposure. In the present study, we present a novel immunogen to selectively induce CD8(+) T cells but not CD4(+) T cells targeting viral antigens. The immunogen, CaV11, was constructed by tandem connection of overlapping 11-mer peptides spanning simian immunodeficiency virus (SIV) Gag capsid (CA) and Vif. Prime-boost immunization with DNA and Sendai virus (SeV) vectors expressing CaV11 efficiently induced Gag/Vif-specific CD8(+) T-cell responses with inefficient Gag/Vif-specific CD4(+) T-cell induction in rhesus macaques (n = 6). None of the macaques exhibited the enhancement of acute viral replication after an intravenous high-dose SIV challenge, which was observed in those immunized with DNA and SeV expressing the whole Gag protein in our previous study. Set point viral control postinfection was associated with SeV-specific CD4(+) T-cell responses postimmunization, suggesting contribution of SeV-specific helper responses to effective Gag/Vif-specific CD8(+) T-cell induction by vaccination. This immunogen design could be a promising method for selective induction of effective anti-HIV CD8(+) T-cell responses. IMPORTANCE Induction of effective CD8(+) T-cell responses is an important HIV vaccine strategy. Several promising vaccine delivery tools have been developed, and immunogen optimization is now crucial for effective T-cell induction. Conventional immunogens have been designed to induce virus-specific CD4(+) T cells as well as CD8(+) T cells, but induction of virus-specific CD4(+) T cells that are preferential targets for HIV infection could enhance acute HIV proliferation. Here, we designed a novel immunogen to induce HIV-specific CD8(+) T cells without HIV-specific CD4(+) T-cell induction but with non-HIV antigen-specific CD4(+) T-cell help. Our analysis in a macaque AIDS model showed that our immunogen can efficiently elicit effective CD8(+) T but not CD4(+) T cells targeting viral antigens, resulting in no enhancement of acute viral replication after virus exposure. This immunogen design, also applicable for other currently developed immunogens, could be a promising method for selective induction of effective anti-HIV CD8(+) T-cell responses.

CD8+ T-cell responses exert strong suppressive pressure on HIV replication and select for viral escape mutations. Some of these major histocompatibility complex class I (MHC-I)-associated mutations result in reduction of in vitro viral replicative capacity. While these mutations can revert after viral transmission to MHC-I-disparate hosts, recent studies have suggested that these MHC-I-associated mutations accumulate in populations and make viruses less pathogenic in vitro. Here, we directly show an increase in the in vivo virulence of an MHC-I-adapted virus serially-passaged through MHC-I-mismatched hosts in a macaque AIDS model despite a reduction in in vitro viral fitness. The first passage simian immunodeficiency virus (1pSIV) obtained 1 year after SIVmac239 infection in a macaque possessing a protective MHC-I haplotype 90-120-Ia was transmitted into 90-120-Ia- macaques, whose plasma 1 year post-infection was transmitted into other 90-120-Ia- macaques to obtain the third passage SIV (3pSIV). Most of the 90-120-Ia-associated mutations selected in 1pSIV did not revert even in 3pSIV. 3pSIV showed lower in vitro viral fitness but induced persistent viremia in 90-120-Ia- macaques. Remarkably, 3pSIV infection in 90-120-Ia+ macaques resulted in significantly higher viral loads and reduced survival compared to wild-type SIVmac239. These results indicate that MHC-I-adapted SIVs serially-transmitted through MHC-I-mismatched hosts can have higher virulence in MHC-I-matched hosts despite their lower in vitro viral fitness. This study suggests that multiply-passaged HIVs could result in loss of HIV-specific CD8+ T cell responses in human populations and the in vivo pathogenic potential of these escaped viruses may be enhanced., Author summary CD8+ T-cell responses exert considerable control over replication of HIV and select for viral escape mutations. Recent studies have suggested that these major histocompatibility complex class I (MHC-I)-associated mutations accumulate in populations and make viruses less pathogenic in vitro. Other studies have shown that some of these escape mutations can revert after passage to MHC-I-disparate hosts. In an attempt to reconcile these apparently conflicting results, we serially passaged a virus isolate through MHC-I-mismatched hosts in the macaque AIDS model of simian immunodeficiency virus (SIV) infection. Here we show an increase in the in vivo virulence of an MHC-I-adapted virus despite a reduction in in vitro viral replication capacity. Only a few of the selected escape mutations reverted after transmission to MHC-I-disparate recipients. Results clearly showed that MHC-I-adapted SIVs that have been serially-transmitted through MHC-I-mismatched hosts can have higher in vivo virulence in MHC-I-matched hosts despite their lower in vitro viral fitness. This study suggests that HIVs may become less sensitive to CD8+ T cell responses and could have increased in vivo virulence by adaptation to MHC-I in human populations.

CD4 + T-cell responses are crucial for effective antibody and CD8 + T-cell induction following virus infection. However, virus-specific CD4 + T cells can be preferential targets for human immunodeficiency virus (HIV) infection. HIV-specific CD4 + T-cell induction by vaccination may thus result in enhancement of virus replication following infection. In the present study, we show that vaccine-elicited CD4 + T cells expressing CD107a are relatively resistant to depletion in a macaque AIDS model. Comparison of virus-specific CD107a, macrophage inflammatory protein-1β, gamma interferon, tumor necrosis factor alpha, and interleukin-2 responses in CD4 + T cells of vaccinated macaques prechallenge and 1 week postchallenge showed a significant reduction in the CD107a − but not the CD107a + subset after virus exposure. Those vaccinees that failed to control viremia showed a more marked reduction and exhibited significantly higher viral loads at week 1 than unvaccinated animals. Our results indicate that vaccine-induced CD107a − CD4 + T cells are depleted following virus infection, suggesting a rationale for avoiding virus-specific CD107a − CD4 + T-cell induction in HIV vaccine design. IMPORTANCE Induction of effective antibody and/or CD8 + T-cell responses is a principal vaccine strategy against human immunodeficiency virus (HIV) infection. CD4 + T-cell responses are crucial for effective antibody and CD8 + T-cell induction. However, virus-specific CD4 + T cells can be preferential targets for HIV infection. Here, we show that vaccine-induced virus-specific CD107a − CD4 + T cells are largely depleted following infection in a macaque AIDS model. While CD4 + T-cell responses are important in viral control, our results indicate that virus-specific CD107a − CD4 + T-cell induction by vaccination may not lead to efficient CD4 + T-cell responses following infection but rather be detrimental and accelerate viral replication in the acute phase. This suggests that HIV vaccine design should avoid virus-specific CD107a − CD4 + T-cell induction. Conversely, this study found that vaccine-induced CD107a + CD4 + T cells are relatively resistant to depletion following virus challenge, implying that induction of these cells may be an alternative approach toward HIV control.

Virus-specific CD8+ T cells exert strong suppressive pressure on human/simian immunodeficiency virus (HIV/SIV) replication. These responses have been intensively examined in peripheral blood mononuclear cells (PBMCs) but not fully analyzed in lymph nodes (LNs), where interaction between CD8+ T cells and HIV/SIV-infected cells occurs. Here, we investigated target antigen specificity of CD8+ T cells in LNs in a macaque AIDS model. Analysis of virus antigen-specific CD8+ T-cell responses in the inguinal LNs obtained from twenty rhesus macaques in the chronic phase of SIV infection showed an inverse correlation between viral loads and frequencies of CD8+ T cells with CD28+ CD95+ central memory phenotype targeting the N-terminal half of SIV core antigen (Gag-N). In contrast, analysis of LNs but not PBMCs revealed a positive correlation between viral loads and frequencies of CD8+ T cells with CD28−CD95+ effector memory phenotype targeting the N-terminal half of SIV envelope (Env-N), soluble antigen. Indeed, LNs with detectable SIV capsid p27 antigen in the germinal center exhibited significantly lower Gag-N-specific CD28+ CD95+ CD8+ T-cell and higher Env-N-specific CD28−CD95+ CD8+ T-cell responses than those without detectable p27. These results imply that core and envelope antigen-specific CD8+ T cells show different patterns of interactions with HIV/SIV-infected cells.

Identifying human immunodeficiency virus type 1 (HIV-1) control mechanisms by neutralizing antibodies (NAbs) is critical for anti-HIV-1 strategies. Recent in vivo studies on animals infected with simian immunodeficiency virus (SIV) and related viruses have shown the efficacy of postinfection NAb passive immunization for viremia reduction, and one suggested mechanism is its occurrence through modulation of cellular immune responses. Here, we describe SIV control in macaques showing biphasic CD8 + cytotoxic T lymphocyte (CTL) responses following acute-phase NAb passive immunization. Analysis of four SIV mac239 -infected rhesus macaque pairs matched with major histocompatibility complex class I haplotypes found that counterparts receiving day 7 anti-SIV polyclonal NAb infusion all suppressed viremia for up to 2 years without accumulating viral CTL escape mutations. In the first phase of primary viremia control attainment, CD8 + cells had high capacities to suppress SIVs carrying CTL escape mutations. Conversely, in the second, sustained phase of SIV control, CTL responses converged on a pattern of immunodominant CTL preservation. During this sustained phase of viral control, SIV epitope-specific CTLs showed retention of phosphorylated extracellular signal-related kinase (ERK) hi /phosphorylated AMP-activated protein kinase (AMPK) lo subpopulations, implying their correlation with SIV control. The results suggest that virus-specific CTLs functionally boosted by acute-phase NAbs may drive robust AIDS virus control. IMPORTANCE In early HIV infection, NAb responses are lacking and CTL responses are insufficient, which leads to viral persistence. Hence, it is important to identify immune responses that can successfully control such HIV replication. Here, we show that monkeys receiving NAb passive immunization in early SIV infection strictly control viral replication for years. Passive infusion of NAbs with CTL cross-priming capacity resulted in induction of functionally boosted early CTL responses showing enhanced suppression of CTL escape mutant virus replication. Accordingly, the NAb-infused animals did not show accumulation of viral CTL escape mutations during sustained SIV control, and immunodominant CTL responses were preserved. This early functional augmentation of CTLs by NAbs provides key insights into the design of lasting and viral escape mutation-free protective immunity against HIV-1 infection.

Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious disease of pigs. There are numerous CSFV strains that differ in virulence, resulting in clinical disease with different degrees of severity. Low-virulent and moderately virulent isolates cause a mild and often chronic disease, while highly virulent isolates cause an acute and mostly lethal hemorrhagic fever. The live attenuated vaccine strain GPE − was produced by multiple passages of the virulent ALD strain in cells of swine, bovine, and guinea pig origin. With the aim of identifying the determinants responsible for the attenuation, the GPE − vaccine virus was readapted to pigs by serial passages of infected tonsil homogenates until prolonged viremia and typical signs of CSF were observed. The GPE − /P-11 virus isolated from the tonsils after the 11th passage in vivo had acquired 3 amino acid substitutions in E2 (T830A) and NS4B (V2475A and A2563V) compared with the virus before passages. Experimental infection of pigs with the mutants reconstructed by reverse genetics confirmed that these amino acid substitutions were responsible for the acquisition of pathogenicity. Studies in vitro indicated that the substitution in E2 influenced virus spreading and that the changes in NS4B enhanced the viral RNA replication. In conclusion, the present study identified residues in E2 and NS4B of CSFV that can act synergistically to influence virus replication efficiency in vitro and pathogenicity in pigs.

Nonhuman primate AIDS models are essential for the analysis of AIDS pathogenesis and the evaluation of vaccine efficacy. Multiple studies on human immunodeficiency virus and simian immunodeficiency virus (SIV) infection have indicated the association of major histocompatibility complex class I (MHC-I) genotypes with rapid or slow AIDS progression. The accumulation of macaque groups that share not only a single MHC-I allele but also an MHC-I haplotype consisting of multiple polymorphic MHC-I loci would greatly contribute to the progress of AIDS research. Here, we investigated SIVmac239 infections in four groups of Burmese rhesus macaques sharing individual MHC-I haplotypes, referred to as A, E, B, and J. Out of 20 macaques belonging to A + ( n = 6), E + ( n = 6), B + ( n = 4), and J + ( n = 4) groups, 18 showed persistent viremia. Fifteen of them developed AIDS in 0.5 to 4 years, with the remaining three at 1 or 2 years under observation. A + animals, including two controllers, showed slower disease progression, whereas J + animals exhibited rapid progression. E + and B + animals showed intermediate plasma viral loads and survival periods. Gag-specific CD8 + T-cell responses were efficiently induced in A + animals, while Nef-specific CD8 + T-cell responses were in A + , E + , and B + animals. Multiple comparisons among these groups revealed significant differences in survival periods, peripheral CD4 + T-cell decline, and SIV-specific CD4 + T-cell polyfunctionality in the chronic phase. This study indicates the association of MHC-I haplotypes with AIDS progression and presents an AIDS model facilitating the analysis of virus-host immune interaction.

In our previous study, we genetically analyzed bovine viral diarrhea viruses (BVDVs) isolated from 2000 to 2006 in Japan and reported that subgenotype 1b viruses were predominant. In the present study, 766 BVDVs isolated from 2006 to 2014 in Hokkaido, Japan, were genetically analyzed to understand recent epidemics. Phylogenetic analysis based on nucleotide sequences of the 5′-untranslated region of viral genome revealed that 766 isolates were classified as genotype 1 (BVDV-1; 544 isolates) and genotype 2 (BVDV-2; 222). BVDV-1 isolates were further divided into BVDV-1a (93), 1b (371) and 1c (80) subgenotypes, and all BVDV-2 isolates were grouped into BVDV-2a subgenotype (222). Further comparative analysis was performed with BVDV-1a, 1b and 2a viruses isolated from 2001 to 2014. Phylogenetic analysis based on nucleotide sequences of the viral glycoprotein E2 gene, a major target of neutralizing antibodies, revealed that BVDV-1a, 1b and 2a isolates were further classified into several clusters. Cross-neutralization tests showed that BVDV-1b isolates were antigenically different from BVDV-1a isolates, and almost BVDV-1a, 1b and 2a isolates were antigenically similar among each subgenotype and each E2 cluster. Taken together, BVDV-1b viruses are still predominant, and BVDV-2a viruses have increased recently in Hokkaido, Japan. Field isolates of BVDV-1a, 1b and 2a show genetic diversity on the E2 gene with antigenic conservation among each subgenotype during the last 14 years.

Control of HIV replication is a rare immunological event, providing clues to understand the viral control mechanism. CD8+ T-cell responses are crucial for virus control, but it is unclear whether lasting HIV containment can be achieved after establishment of infection. Here, we describe lasting SIV containment in a macaque AIDS model. Analysis of ten rhesus macaques that controlled viremia for 2 years post-infection found accumulation of proviral gag and nef CD8+ T-cell escape mutations in four of them. These four controllers mounted CD8+ T cells targeting Gag, Nef, and other viral proteins at 4 months, suggesting that broadening of CD8+ T-cell targets can be an indicator of the beginning of viral control failure. The remaining six aviremic SIV controllers, however, harbored proviruses without mutations and showed no or little broadening of their CD8+ T-cell responses in the chronic phase. Indeed, three of the latter six exhibiting no change in CD8+ T-cell targets showed gradual decreases in SIV-specific CD8+ T-cell frequencies, implying a concomitant reduction in viral replication. Thus, stability of the breadth of virus-specific CD8+ T-cell responses may represent a status of lasting HIV containment by CD8+ T cells., Author Summary CD8+ T-cell responses are crucial for HIV control, but it is unclear whether lasting HIV containment can be achieved after establishment of infection. Several T cell-based vaccine trials have currently shown primary viremia control in macaque AIDS models of simian immunodeficiency virus (SIV) infection, but residual viral replication may occur, followed by accumulation of viral CD8+ T-cell escape mutations, possibly leading to eventual viremia rebound. In the present study, we analyzed ten rhesus macaques that controlled SIV replication without detectable viremia for more than 2 years. Animals were divided into two groups on the basis of proviral genome sequences at 2 years post-infection. Analysis of the first group exhibiting multiple CD8+ T-cell escape mutations indicated that broadening of CD8+ T-cell responses can be an indicator of the beginning of viral control failure. Conversely, analysis of the second group having no mutation suggested that stability of the breadth of virus-specific CD8+ T-cell responses represents a status of lasting HIV containment by CD8+ T cells. Thus, this study presents a model of stable SIV containment, contributing to elucidation of the requisites for lasting HIV control.

For development of an effective T cell-based AIDS vaccine, it is critical to define the antigens that elicit the most potent responses. Recent studies have suggested that Gag-specific and possibly Vif/Nef-specific CD8 + T cells can be important in control of the AIDS virus. Here, we tested whether induction of these CD8 + T cells by prophylactic vaccination can result in control of simian immunodeficiency virus (SIV) replication in Burmese rhesus macaques sharing the major histocompatibility complex class I (MHC-I) haplotype 90-010-Ie associated with dominant Nef-specific CD8 + T-cell responses. In the first group vaccinated with Gag-expressing vectors ( n = 5 animals), three animals that showed efficient Gag-specific CD8 + T-cell responses in the acute phase postchallenge controlled SIV replication. In the second group vaccinated with Vif- and Nef-expressing vectors ( n = 6 animals), three animals that elicited Vif-specific CD8 + T-cell responses in the acute phase showed SIV control, whereas the remaining three with Nef-specific but not Vif-specific CD8 + T-cell responses failed to control SIV replication. Analysis of 18 animals, consisting of seven unvaccinated noncontrollers and the 11 vaccinees described above, revealed that the sum of Gag- and Vif-specific CD8 + T-cell frequencies in the acute phase was inversely correlated with plasma viral loads in the chronic phase. Our results suggest that replication of the AIDS virus can be controlled by vaccine-induced subdominant Gag/Vif epitope-specific CD8 + T cells, providing a rationale for the induction of Gag- and/or Vif-specific CD8 + T-cell responses by prophylactic AIDS vaccines.

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for a screening test to detect antibodies against classical swine fever virus (CSFV). Viral glycoproteins, which were purified from swine kidney cells infected with CSFV ALD/A76 strain by the immunoaffinity purification using monoclonal antibody against E2 protein, were adsorbed on a microtiter plate as the antigen for the antibody detection. Each antibody titer of serum sample was expressed as a sample per positive value calculated with optical absorbance of each sample and that of a positive control. The advantage of this ELISA is its higher sensitivity: most sera containing more than 4 neutralization titers were determined to be positive. This ELISA is unable to discriminate between antibodies against CSFV and those against other ruminant pestiviruses, therefore positive sera in this ELISA should be evaluated by a cross-neutralization test using CSFV, bovine viral diarrhea virus, and border disease virus. Taken together, the indirect ELISA developed in this study is useful screening tool to detect antibodies against CSFV for the large-scale monitoring of classical swine fever.

Virus-specific cytotoxic T lymphocytes (CTLs) are major effectors in acquired immune responses against viral infection. Virus-specific CTLs recognize specific viral peptides presented by major histocompatibility complex class-I (MHC-I) on the surface of virus-infected target cells via their T cell receptor (TCR) and eliminate target cells by both direct and indirect mechanisms. In human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections, host immune responses fail to contain the virus and allow persistent viral replication, leading to AIDS progression. CTL responses exert strong suppressive pressure on HIV/SIV replication and cumulative studies have indicated association of HLA/MHC-I genotypes with rapid or slow AIDS progression.

Induction of durable cellular immune responses by vaccination is an important strategy for the control of persistent pathogen infection. Viral vectors are promising vaccine tools for eliciting antigen-specific T-cell responses. Repeated vaccination may contribute to durable memory T-cell induction, but anti-vector antibodies could be an obstacle to its efficacy. We previously developed a Sendai virus (SeV) vector vaccine and showed the potential of this vector for efficient T-cell induction in macaques. Here, we examined whether repeated SeV vector vaccination with short intervals can enhance antigen-specific CD8(+) T-cell responses. Four rhesus macaques possessing the MHC-I haplotype 90-120-Ia were immunized three times with intervals of three weeks. For the vaccination, we used replication-defective F-deleted SeV vectors inducing CD8(+) T-cell responses specific for simian immunodeficiency virus Gag(206-216) and Gag(241-249), which are dominant epitopes restricted by 90-120-Ia-derived MHC-I molecules. All four animals showed higher Gag(206-216)-specific and Gag(241-249)-specific CD8(+) T-cell responses after the third vaccination than those after the first vaccination, indicating enhancement of antigen-specific CD8(+) T-cell responses by the second/third SeV vector vaccination even with short intervals. These results suggest that repeated SeV vector vaccination can contribute to induction of efficient and durable T-cell responses.

In the natural courses of human immunodeficiency virus (HIV) infections, host immune responses fail to contain the virus and allow persistent HIV replication, leading to AIDS progression. For development of an effective vaccine against those viral infections which do not show spontaneous remission, it is important to elucidate which immune responses to be induced for viral control. This review focuses on antibodies and cytotoxic T lymphocytes, key adaptive immune effectors, and discusses possible mechanisms for HIV control by vaccine-induced antibody, memory B lymphocyte, and (effector and central) memory T lymphocyte responses. Finally, we mention the ongoing international project for a clinical trial of our Sendai virus vector-based AIDS vaccine.

Background Optimization of antigens as well as delivery system is crucial for development of an effective T-cell based AIDS vaccine. Our recent results suggested higher antiviral efficacy of Vifand Nef-specific CTLs as well as Gag-specific ones (JEM 199:1709, 2004; AIDS 24:2777, 2010). Here, we examined efficacy of Gag-specific or Vif/Nef-specific CTL induction by vaccination against SIV infection.