Enhancement of IL-6 Production Induced by SARS-CoV-2 Nucleocapsid Protein and Bangladeshi COVID-19 Patients' Sera.

Coronavirus disease 2019 (COVID-19) is a respiratory tract infection caused by severe acute respiratory syndrome coronavirus 2 that can have detrimental effects on multiple organs and accelerate patient mortality. This study, which encompassed 130 confirmed COVID-19 patients who were assessed at three different time points (i.e., 3, 7, and 12 days) after the onset of symptoms, investigated interleukin-6 (IL-6) enhancement induced by a viral nucleocapsid (N) protein from a myeloid cell line. Disease severity was categorized as mild, moderate, or severe. The severe cases were characterized as having significant elevations in serum IL-6, C-reactive protein, D-dimer, ferritin, creatinine, leukocytes, and neutrophil-to-lymphocyte ratio and decreased hemoglobin, hematocrit, and albumin levels compared with mild and moderate cases. To evaluate IL-6-inducing activity, heat-inactivated sera from these patients were incubated with and without the N protein. The findings showed a progressive increase in IL-6 production in severe cases upon N protein stimulation. There was a strong correlation between anti-N antibodies and levels of IL-6 secreted by myeloid cells in the presence of N protein and sera, indicating the crucial role that the anti-N antibody plays in inducing IL-6 production. Uncontrolled IL-6 production played a pivotal role in disease pathogenesis, exacerbating both disease severity and mortality. Efficiently targeting the N protein could potentially be employed as a therapeutic strategy for regulating the immune response and alleviating inflammation in severe cases.

Anti-nucleocapsid antibodies enhance the production of IL-6 induced by SARS-CoV-2 N protein.

A cytokine storm induces acute respiratory distress syndrome, the main cause of death in coronavirus disease 2019 (COVID-19) patients. However, the detailed mechanisms of cytokine induction due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain unclear. To examine the cytokine production in COVID-19, we mimicked the disease in SARS-CoV-2-infected alveoli by adding the lysate of SARS-CoV-2-infected cells to cultured macrophages or induced pluripotent stem cell-derived myeloid cells. The cells secreted interleukin (IL)-6 after the addition of SARS-CoV-2-infected cell lysate. Screening of 25 SARS-CoV-2 protein-expressing plasmids revealed that the N protein-coding plasmid alone induced IL-6 production. The addition of anti-N antibody further enhanced IL-6 production, but the F(ab')2 fragment did not. Sera from COVID-19 patients also enhanced IL-6 production, and sera from patients with severer disease induced higher levels of IL-6. These results suggest that anti-N antibody promotes IL-6 production in SARS-CoV-2-infected alveoli, leading to the cytokine storm of COVID-19.

The potential of COVID-19 patients' sera to cause antibody-dependent enhancement of infection and IL-6 production.

Since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), many vaccine trials have been initiated. An important goal of vaccination is the development of neutralizing antibody (Ab) against SARS-CoV-2. However, the possible induction of antibody-dependent enhancement (ADE) of infection, which is known for other coronaviruses and dengue virus infections, is a particular concern in vaccine development. Here, we demonstrated that human iPS cell-derived, immortalized, and ACE2- and TMPRSS2-expressing myeloid cell lines are useful as host cells for SARS-CoV-2 infection. The established cell lines were cloned and screened based on their function in terms of susceptibility to SARS-CoV-2-infection or IL-6 productivity. Using the resulting K-ML2 (AT) clone 35 for SARS-CoV-2-infection or its subclone 35-40 for IL-6 productivity, it was possible to evaluate the potential of sera from severe COVID-19 patients to cause ADE and to stimulate IL-6 production upon infection with SARS-CoV-2.

Generation of macrophages with altered viral sensitivity from genome-edited rhesus macaque iPSCs to model human disease.

Because of their close biological similarity to humans, non-human primate (NHP) models are very useful for the development of induced pluripotent stem cell (iPSC)-based cell and regenerative organ transplantation therapies. However, knowledge on the establishment, differentiation, and genetic modification of NHP-iPSCs, especially rhesus macaque iPSCs, is limited. We succeeded in establishing iPSCs from the peripheral blood of rhesus macaques (Rh-iPSCs) by combining the Yamanaka reprograming factors and two inhibitors (GSK-3 inhibitor [CHIR 99021] and MEK1/2 inhibitor [PD0325901]) and differentiated the cells into functional macrophages through hematopoietic progenitor cells. To confirm feasibility of the Rh-iPSC-derived macrophages as a platform for bioassays to model diseases, we knocked out TRIM5 gene in Rh-iPSCs by CRISPR-Cas9, which is a species-specific HIV resistance factor. TRIM5 knockout (KO) iPSCs had the same differentiation potential to macrophages as did Rh-iPSCs, but the differentiated macrophages showed a gain of sensitivity to HIV infection in vitro. Our reprogramming, gene editing, and differentiation protocols used to obtain Rh-iPSC-derived macrophages can be applied to other gene mutations, expanding the number of NHP gene therapy models.

A Novel Sub-Lineage of Chikungunya Virus East/Central/South African Genotype Indian Ocean Lineage Caused Sequential Outbreaks in Bangladesh and Thailand.

In recent decades, chikungunya virus (CHIKV) has become geographically widespread. In 2004, the CHIKV East/Central/South African (ECSA) genotype moved from Africa to Indian ocean islands and India followed by a large epidemic in Southeast Asia. In 2013, the CHIKV Asian genotype drove an outbreak in the Americas. Since 2016, CHIKV has re-emerged in the Indian subcontinent and Southeast Asia. In the present study, CHIKVs were obtained from Bangladesh in 2017 and Thailand in 2019, and their nearly full genomes were sequenced. Phylogenetic analysis revealed that the recent CHIKVs were of Indian Ocean Lineage (IOL) of genotype ECSA, similar to the previous outbreak. However, these CHIKVs were all clustered into a new distinct sub-lineage apart from the past IOL CHIKVs, and they lacked an alanine-to-valine substitution at position 226 of the E1 envelope glycoprotein, which enhances CHIKV replication in Aedes albopictus. Instead, all the re-emerged CHIKVs possessed mutations of lysine-to-glutamic acid at position 211 of E1 and valine-to-alanine at position 264 of E2. Molecular clock analysis suggested that the new sub-lineage CHIKV was introduced to Bangladesh around late 2015 and Thailand in early 2017. These results suggest that re-emerged CHIKVs have acquired different adaptations than the previous CHIKVs.

Promising application of monoclonal antibody against chikungunya virus E1-antigen across genotypes in immunochromatographic rapid diagnostic tests.

BACKGROUND: Three different genotypes of chikungunya virus (CHIKV) have been classified: East/Central/South African (ECSA), West African (WA), and Asian. Previously, a rapid immunochromatographic (IC) test detecting CHIKV E1-antigen showed high sensitivity for certain ECSA-genotype viruses, but this test showed poor performance against the Asian-genotype virus that is spreading in the American continents. We found that the reactivity of one monoclonal antibody (MAb) used in the IC rapid diagnostic test (RDT) is affected by a single amino acid substitution in E1. Therefore, we developed new MAbs that exhibited specific recognition of all three genotypes of CHIKV. METHODS: Using a combination of the newly generated MAbs, we developed a novel version of the IC RDT with improved sensitivity to Asian-genotype CHIKV. To evaluate the sensitivity, specificity, and cross-reactivity of the new version of the IC RDT, we first used CHIKV isolates and E1-pseudotyped lentiviral vectors. We then used clinical specimens obtained in Aruba in 2015 and in Bangladesh in 2017 for further evaluation of RDT sensitivity and specificity. Another alphavirus, sindbis virus (SINV), was used to test RDT cross-reactivity. RESULTS: The new version of the RDT detected Asian-genotype CHIKV at titers as low as 10^4 plaque-forming units per mL, a concentration that was below the limit of detection of the old version. The new RDT had sensitivity to the ECSA genotype that was comparable with that of the old version, yielding 92% (92 out of 100) sensitivity (95% confidence interval 85.0-95.9) and 100% (100 out of 100) specificity against a panel of 100 CHIKV-positive and 100 CHIKV-negative patient sera obtained in the 2017 outbreak in Bangladesh. CONCLUSIONS: Our newly developed CHIKV antigen-detecting RDT demonstrated high levels of sensitivity and lacked cross-reactivity against SINV. These results suggested that our new version of the CHIKV E1-antigen RDT is promising for use in areas in which the Asian and ECSA genotypes of CHIKV circulate. Further validation with large numbers of CHIKV-positive and -negative clinical samples is warranted. (323 words).

Genotype replacement of dengue virus type 3 and clade replacement of dengue virus type 2 genotype Cosmopolitan in Dhaka, Bangladesh in 2017.

Dengue is a mosquito-borne disease that has spread to >100 countries and is caused by the dengue virus (DENV), which belongs to the Flavivirus genus of the family Flaviviridae. DENV comprises 4 serotypes (DENV-1 to -4), and each serotype is further divided into distinct genotypes. In India, it is reported that all 4 serotypes of DENV co-circulate. Although Bangladesh is a neighboring country of India, very few reports have published DENV sequence data for the country, especially after 2012. To understand the current distribution of DENV genotypes in Bangladesh, we determined the nucleotide sequences of envelope regions obtained from 58 DENV-positive patients diagnosed at Apollo Hospitals Dhaka during the period between September 2017 and February 2018. We found 5 DENV-1, 47 DENV-2, and 6 DENV-3 serotypes. A phylogenetic analysis of the obtained viral sequences revealed that DENV-3 genotype I was present instead of DENV-3 genotype II, which was predominant in Bangladesh between 2000 and 2009. Furthermore, we found two distinct lineages of the Cosmopolitan genotype of DENV-2, one of which was closely related to strains from Southeast Asia and has never been reported previously in Bangladesh. These results indicated that DENVs in Bangladesh have increased in genotypic diversity and suggest that the DENV genotypic shift observed in other Asian countries also might have been taking place in Bangladesh.

Variation at position 350 in the Chikungunya virus 6K-E1 protein determines the sensitivity of detection in a rapid E1-antigen test.

Chikungunya virus (CHIKV), a mosquito-borne pathogen, consists of three genotypes: East/Central/South African (ECSA), West African (WA), and Asian. Although a current rapid immunochromatographic (IC) test detecting CHIKV E1-antigen showed high sensitivity to ECSA-genotype viruses, it showed poor performance against the Asian-genotype virus that is spreading in the American continents. To understand the basis for the low performance of this IC test against Asian-genotype virus, we re-examined the anti-CHIKV monoclonal antibodies (mAbs) used in the assay for their interaction with E1-antigen of the three CHIKV genotypes. We found that the reactivity of one mAb for Asian-genotype virus was lower than that for ECSA virus. Comparison of E1 amino acid sequences revealed that the ECSA virus used to generate these mAbs possesses glutamic acid (E) at position 350, in contrast to WA and Asian, which possess aspartic acid (D) at this position. Site-directed mutagenesis confirmed that the mutation altered mAb reactivity, since E-to-D substitution at position 350 in ECSA reduced recognition by the mAb, while D-to-E substitution at this position in Asian and WA increased affinity for the mAb. Taken together, these results indicate that residue 350 of the CHIKV 6K-E1 is a key element affecting the performance of this IC assay.

A Single-Nucleotide Polymorphism in ABCC4 Is Associated with Tenofovir-Related Beta2-Microglobulinuria in Thai Patients with HIV-1 Infection.

BACKGROUND: In Thailand, the combined generic anti-retroviral drug stavudine/lamivudine/nevirapine (d4T/3TC/NVP) has been used to treat human immunodeficiency virus (HIV)-infected individuals since 2001. Due to relatively frequent adverse effects, d4T gradually has been replaced with tenofovir disoproxil fumarate (TDF). Although the frequency of adverse drug effects with TDF is lower than that with d4T, TDF is known to induce kidney dysfunction, especially in the proximal tubules. It has been reported that renal tubular transporters, including members of the multi-drug resistant (MDR) protein family, are implicated in tenofovir extrusion and may, therefore, confer susceptibility to TDF-induced kidney tubular dysfunction (KTD). We have explored the association between KTD and polymorphisms in genes that encode adenosine triphosphate-binding cassette (ABC)-type MDRs. METHODS: HIV-infected patients receiving TDF-containing antiretroviral regimens for at least one year were enrolled in the study. The levels of beta2-microglobulin in urine and creatinine (Cr) were measured. Three single-nucleotide polymorphisms, ABCC2 C-24T (rs717620), ABCC2 G1429A (rs2273697), and ABCC4 T4976C (rs1059751), were analyzed using TaqMan SNP genotyping assays. RESULTS: A total of 273 HIV-infected patients were recruited. The median number of years of TDF treatment was 5.04 with interquartile range (IQR) of 3.9-6.7. Despite the length of treatment with TDF, 98.5% patients maintained an estimated glomerular filtration rate (eGFR) of >60 mL/min as calculated by the CKD-EPI formula. Fifty-four patients (19.8%) showed beta2-microglobulinuria (median 2636 µg/g Cr with IQR of 1519-13197 µg/g Cr). The allele frequency of ABCC4 T4976C among those 54 patients was 0.602, compared to 0.475 among the 219 remaining patients (p = 0.018). CONCLUSIONS: Approximately 20% of HIV-infected patients receiving TDF showed beta2-microglobulinuria. The C allele at position 4976 of the ABCC4 gene was associated with beta2-microglobulinuria in this population. This polymorphism may help to identify patients at greater risk for developing TDF-associated KTD.

Moderate restriction of macrophage-tropic human immunodeficiency virus type 1 by SAMHD1 in monocyte-derived macrophages.

Macrophage-tropic human immunodeficiency virus type 1 (HIV-1) strains are able to grow to high titers in human monocyte-derived macrophages. However, it was recently reported that cellular protein SAMHD1 restricts HIV-1 replication in human cells of the myeloid lineage, including monocyte-derived macrophages. Here we show that degradation of SAMHD1 in monocyte-derived macrophages was associated with moderately enhanced growth of the macrophage-tropic HIV-1 strain. SAMHD1 degradation was induced by treating target macrophages with vesicular stomatitis virus glycoprotein-pseudotyped human immunodeficiency virus type 2 (HIV-2) particles containing viral protein X. For undifferentiated monocytes, HIV-2 particle treatment allowed undifferentiated monocytes to be fully permissive for productive infection by the macrophage-tropic HIV-1 strain. In contrast, untreated monocytes were totally resistant to HIV-1 replication. These results indicated that SAMHD1 moderately restricts even a macrophage-tropic HIV-1 strain in monocyte-derived macrophages, whereas the protein potently restricts HIV-1 replication in undifferentiated monocytes.

Generation of rhesus macaque-tropic HIV-1 clones that are resistant to major anti-HIV-1 restriction factors.

Human immunodeficiency virus type 1 (HIV-1) replication in macaque cells is restricted mainly by antiviral cellular APOBEC3, TRIM5α/TRIM5CypA, and tetherin proteins. For basic and clinical HIV-1/AIDS studies, efforts to construct macaque-tropic HIV-1 (HIV-1mt) have been made by us and others. Although rhesus macaques are commonly and successfully used as infection models, no HIV-1 derivatives suitable for in vivo rhesus research are available to date. In this study, to obtain novel HIV-1mt clones that are resistant to major restriction factors, we altered Gag and Vpu of our best HIV-1mt clone described previously. First, by sequence- and structure-guided mutagenesis, three amino acid residues in Gag-capsid (CA) (M94L/R98S/G114Q) were found to be responsible for viral growth enhancement in a macaque cell line. Results of in vitro TRIM5α susceptibility testing of HIV-1mt carrying these substitutions correlated well with the increased viral replication potential in macaque peripheral blood mononuclear cells (PBMCs) with different TRIM5 alleles, suggesting that the three amino acids in HIV-1mt CA are involved in the interaction with TRIM5α. Second, we replaced the transmembrane domain of Vpu of this clone with the corresponding region of simian immunodeficiency virus SIVgsn166 Vpu. The resultant clone, MN4/LSDQgtu, was able to antagonize macaque but not human tetherin, and its Vpu effectively functioned during viral replication in a macaque cell line. Notably, MN4/LSDQgtu grew comparably to SIVmac239 and much better than any of our other HIV-1mt clones in rhesus macaque PBMCs. In sum, MN4/LSDQgtu is the first HIV-1 derivative that exhibits resistance to the major restriction factors in rhesus macaque cells.

Polymorphisms in Fas gene is associated with HIV-related lipoatrophy in Thai patients.

The present study aimed to evaluate the role of genetic polymorphisms in the emergence of lipoatrophy or lipodystrophy in HIV-infected patients with antiretroviral therapy (ART) in Thailand. Position 455 upstream of the Apolipoprotein C3 gene (ApoC3 T-455C, rs2854116), codon 64 of the Beta3 adrenergic receptor gene (ARß3 Tcod64C, rs4994), and position 670 upstream of the Fas gene (Fas A-670G, rs1800682) were genotyped in 829 HIV-infected Thai patients who had started ART. Crude and adjusted incidence rate ratios (IRR) were calculated using Poisson regression. The serum levels of cholesterol, triglycerides, high-density lipoprotein (HDL), and low-density lipoprotein (LDL) were also analyzed. Multivariate analysis revealed an association between the Fas -670AA genotype, but not the ApoC3 -455 or ARß3 cod64 genotypes, with the incidence of lipoatrophy after adjusting for gender and stavudine (d4T)-containing regimens (IRR=1.72, 95% CI=1.20-2.45, p=0.003). However, ApoC3 -455C homozygous patients showed elevated serum levels of triglycerides, while this genotype did not affect serum total cholesterol, HDL, or LDL levels in patients with lipoatrophy or lipodystrophy. In contrast, the ARß3 cod64 genotype did not show any significant association with the serum levels of cholesterol, triglycerides, HDL, or LDL. In conclusion, Fas -670AA affected the incidence of lipoatrophy in HIV-1-infected Thai patients, while the ApoC3 -455C allele affected the serum levels of triglycerides. These results confirmed the role of genetics in the development of ART-related metabolic disorders.

Role of Human TRIM5α in Intrinsic Immunity.

Human immunodeficiency virus (HIV) has a very narrow host range. HIV type 1 (HIV-1) does not infect Old World monkeys, such as the rhesus monkey (Rh). Rh TRIM5α was identified as a factor that confers resistance, intrinsic immunity, to HIV-1 infection. Unfortunately, human TRIM5α is almost powerless to restrict HIV-1. However, human TRIM5α potently restricts N-tropic murine leukemia viruses (MLV) but not B-tropic MLV, indicating that human TRIM5α represents the restriction factor previously designated as Ref1. African green monkey TRIM5α represents another restriction factor previously designated as Lv1, which restricts both HIV-1 and simian immunodeficiency virus isolated from macaque (SIVmac) infection. TRIM5 is a member of the tripartite motif family containing RING, B-box2, and coiled-coil domains. The RING domain is frequently found in E3 ubiquitin ligase, and TRIM5α is thought to degrade viral core via ubiquitin-proteasome-dependent and -independent pathways. The alpha isoform of TRIM5 has an additional C-terminal PRYSPRY domain, which is a determinant of species-specific retrovirus restriction by TRIM5α. On the other hand, the target regions of viral capsid protein (CA) are scattered on the surface of core. A single amino acid difference in the surface-exposed loop between α-helices 6 and 7 (L6/7) of HIV type 2 (HIV-2) CA affects viral sensitivity to human TRIM5α and was also shown to be associated with viral load in West African HIV-2 patients, indicating that human TRIM5α is a critical modulator of HIV-2 replication in vivo. Interestingly, L6/7 of CA corresponds to the MLV determinant of sensitivity to mouse factor Fv1, which potently restricts N-tropic MLV. In addition, human genetic polymorphisms also affect antiviral activity of human TRIM5α. Recently, human TRIM5α was shown to activate signaling pathways that lead to activation of NF-κB and AP-1 by interacting with TAK1 complex. TRIM5α is thus involved in control of viral infection in multiple ways.

Impact of glycosylation on antigenicity of simian immunodeficiency virus SIV239: induction of rapid V1/V2-specific non-neutralizing antibody and delayed neutralizing antibody following infection with an attenuated deglycosylated mutant.

Infection of rhesus macaques with a deglycosylation mutant, Delta5G, derived from SIV239, a pathogenic clone of simian immunodeficiency virus (SIV), led to robust acute-phase viral replication followed by a chronic phase with undetectable viral load. This study examined whether humoral responses in Delta5G-infected animals played any role in the control of infection. Neutralizing antibodies (nAbs) were elicited more efficiently in Delta5G-infected animals than in SIV239-infected animals. However, functional nAb measured by 90% neutralization was prominent in only two of the five Delta5G-infected animals, and only at 8 weeks post-infection (p.i.), when viral loads were already below 10(4) copies ml(-1). These results suggest a minimal role for nAbs in the control of the primary infection. In contrast, whilst Ab responses to epitopes localized to the variable loops V1/V2 were detected in all Delta5G-infected animals at 3 weeks p.i., this response was associated with a concomitant reduction in Ab responses to epitopes in gp41 compared with those in SIV239-infected animals. These results suggest that the altered surface glycosylation and/or conformation of viral spikes induce a humoral response against SIV that is distinct from the response induced by SIV239. More interestingly, whereas V1/V2-specific Abs were induced in all animals, these Abs were associated with vigorous Delta5G-specific virion capture ability in only two Delta5G-infected animals that exhibited a functional nAb response. Thus, whereas the deglycosylation mutant infection elicited early virion capture and subsequent nAbs, the responses differed among animals, suggesting the existence of host factors that may influence the functional humoral responses against human immunodeficiency virus/SIV.

Human genetic polymorphisms affecting HIV-1 diseases.

Human immunodeficiency virus type 1 (HIV-1) infection is generally characterized by a long-term, chronic disease course that gradually progresses to acquired immunodeficiency syndrome (AIDS). However, a small fraction of HIV-1-infected individuals remain both clinically and immunologically healthy for 10 years or more after seroconversion. Conversely, the disease of another significant fraction is characterized by an extremely rapid progression to AIDS within 1 year. There are also individuals not infected with HIV-1 who have had repetitive sexual exposure to HIV-1 in extremely high-risk situations. Determining the host factors involved in these different susceptibility and disease courses would be helpful for better understanding AIDS and its control. Today, a worldwide scientific endeavor called the Human Genome Project has completed the production of a full-length sequence of the 3 billion base pairs that make up the human genome. The Human Genome Project has also revealed the presence of variation in human genomes. Relating these genetic differences to human phenotypes offers a very attractive prospect for a genetic understanding of the different sensitivities to various human diseases, including cancer, brain and heart diseases, allergy, and infectious diseases. In this review, we present examples of human genetic variation that can affect HIV-1 infection and disease progression.

Effects of human interleukin 7 on HIV-1 replication in monocyte-derived human macrophages.

Interleukin 7 (IL-7) contributes to development and proliferation of T cells. We investigated the effect of IL-7 on HIV-1 infected monocyte-derived human macrophages. IL-7 treatment of macrophages at a concentration of 10 ng/ml reduced replication of the R5 HIV-1 strain by approximately 50%. Meanwhile, HIV-1-infected macrophages themselves could excrete approximately 20% more IL-7 than uninfected macrophages. These results suggest that IL-7 could be used as a therapeutic modality to recover CD4 T cells.

Influence of glycosylation on the efficacy of an Env-based vaccine against simian immunodeficiency virus SIVmac239 in a macaque AIDS model.

The envelope glycoprotein (Env) of human immunodeficiency viruses (HIVs) and simian immunodeficiency viruses (SIVs) is heavily glycosylated, and this feature has been speculated to be a reason for the insufficient immune control of these viruses by their hosts. In a macaque AIDS model, we demonstrated that quintuple deglycosylation in Env altered a pathogenic virus, SIVmac239, into a novel attenuated mutant virus (delta5G). In delta5G-infected animals, strong protective immunity against SIVmac239 was elicited. These HIV and SIV studies suggested that an understanding of the role of glycosylation is critical in defining not only the virological properties but also the immunogenicity of Env, suggesting that glycosylation in Env could be modified for the development of effective vaccines. To examine the effect of deglycosylation, we constructed prime-boost vaccines consisting of Env from SIVmac239 and delta5G and compared their immunogenicities and vaccine efficacies by challenge infection with SIVmac239. Vaccination-induced immune responses differed between the two vaccine groups. Both Env-specific cellular and humoral responses were higher in wild-type (wt)-Env-immunized animals than in delta5G Env-immunized animals. Following the challenge, viral loads in SIVmac239 Env (wt-Env)-immunized animals were significantly lower than in vector controls, with controlled viral replication in the chronic phase. Unexpectedly, viral loads in delta5G Env-immunized animals were indistinguishable from those in vector controls. This study demonstrated that the prime-boost Env vaccine was effective against homologous SIVmac239 challenge. Changes in glycosylation affected both cell-mediated and humoral immune responses and vaccine efficacy.

[TRIM5alpha].

Human immunodeficiency virus type 1 (HIV-1) shows a very narrow host range limited only to humans and chimpanzees. HIV-1 dose not experimentally infect Old World monkeys, such as rhesus and cynomolgus monkeys, and fails to replicate in activated CD4 positive T lymphocytes obtained from those monkeys. Several lines of evidence have suggested that the block of HIV-1 replication in Old World monkey cells occurred at a post-entry step and appeared to result from a failure to initiate reverse transcription. Recently, the screening of a rhesus monkey cDNA library identified tripartite motif 5 (TRIM5) alpha, a component of cytoplasmic bodies, as a factor that confers resistance to HIV-1 infection. Shortly after, TRIM5alpha of African green monkey, another Old World monkey, was also shown to restrict HIV-1 infection, while human TRIM5alpha was reported to restrict N-tropic murine leukemia virus. Small amino acid differences in the SPRY domain among human and monkey TRIM5alphas were reported to determine species-specific restriction. This review discusses about anti-viral activity of TRIM5alpha.

An efficient and versatile mammalian viral vector system for major histocompatibility complex class I/peptide complexes.

We report a Sendai virus (SeV) vector system for expression of major histocompatibility complex (MHC) class I/peptide complexes. We cloned the extracellular domain of a human MHC class I heavy chain, HLA-A*2402, and human beta-2 microglobulin (beta2m) fused with HLA-A*2402-restricted human immunodeficiency virus type 1 (HIV-1) cytotoxic T-lymphocyte (CTL) epitopes (e-beta2m) in separate SeV vectors. When we coinfected nonhuman mammalian cells with the SeVs, naturally folded human MHC class I/peptide complexes were secreted in the culture supernatants. Biotin binding peptide sequences on the C terminus of the heavy chain were used to tetramerize the complexes. These tetramers made in the SeV system recognized specific CD8-positive T cells in peripheral blood mononuclear cells of HIV-1-positive patients with a specificity and sensitivity similar to those of MHC class I tetramers made in an Escherichia coli system. Solo infection of e-beta2m/SeV produced soluble e-beta2m in the culture supernatant, and cells pulsed with the soluble protein were recognized by specific CTLs. Furthermore, when cells were infected with e-beta2m/SeV, these cells were recognized by the specific CTLs more efficiently than the protein pulse per se. SeV is nonpathogenic for humans, can transduce foreign genes into nondividing cells, and may be useful for immunotherapy to enhance antigen-specific immune responses. Our system can be used not only to detect but also to stimulate antigen-specific cellular immune responses.