Transmission of SARS-CoV-2 during a 2-h domestic flight to Okinawa, Japan, March 2020.

BACKGROUND: Coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus (SARS-CoV-2), has rapidly spread globally. Potentially infected individuals travel on commercial aircraft. Thus, this study aimed to investigate and test the association between the use of face masks, physical distance, and COVID-19 among passengers and flight attendants exposed to a COVID-19 passenger in a domestic flight. METHODS: This observational study investigated passengers and flight attendants exposed to COVID-19 on March 23, 2020, on board a flight to Naha City, Japan. Secondary attack rates were calculated. Whole-genome sequencing of SARS-CoV-2 was used to identify the infectious linkage between confirmed cases in this clustering. The association between confirmed COVID-19 and proximity of passengers' seats to the index case and/or the use of face masks was estimated using logistic regression. RESULTS: Fourteen confirmed and six probable cases were identified among passengers and flight attendants. The secondary attack rate was 9.7%. Twelve of 14 SARS-CoV-2 genome sequences in confirmed cases were identical to that of the index case or showed only one nucleotide mutation. Risk factors for infection included not using a face mask (adjusted odds ratio [aOR]: 7.29, 95% confidence interval [95% CI]: 1.86-28.6), partial face mask use (aOR: 3.0, 95% CI: 0.83-10.8), and being seated within two rows from the index patient (aOR: 7.47, 95% CI: 2.06-27.2). CONCLUSION: SARS-CoV-2 was transmitted on the airplane. Nonuse of face masks was identified as an independent risk factor for contracting COVID-19 on the airplane.

Maternal smoking during pregnancy and rapid weight gain from birth to early infancy.

BACKGROUND: Although several studies have focused on the association between maternal smoking during pregnancy and rapid weight gain (RWG) during infancy, the dose-response relationship has not yet been confirmed, and very few studies have included Asian populations. Using a record-linkage method, we examined the association between maternal smoking during pregnancy and RWG in infants at around 4 months of age to clarify the dose-response relationship. METHODS: Two databases were used: maternal check-ups during pregnancy and early infancy check-ups (between April 1, 2013 and March 31, 2014 in Okinawa, Japan) were linked via IDs and provided to us after unlinkable anonymizing. For 10,433 subjects (5229 boys and 5204 girls), we calculated the change in infants' weight z-score by subtracting the z-score of their birth weight from their weight at early infancy check-ups. Smoking exposure was categorized into five groups. We used Poisson regression to examine the association of maternal smoking during pregnancy with RWG in early infancy. RESULTS: Overall, 1524 (14.6%) were ex-smoker and 511 (4.9%) were current smoker. Compared with the reference category of non-smokers, the adjusted risk ratio of RWG was 1.18 (95% confidence interval [CI], 1.06-1.32) for ex-smokers, 1.18 (95% CI, 0.93-1.50) for those who smoked 1-5 cigarettes per day, 1.57 (95% CI, 1.24-2.00) for those who smoked 6-10 cigarettes per day, and 2.13 (95% CI, 1.51-3.01) for those who smoked ≥11 cigarettes per day. There was a clear dose-response relationship. CONCLUSION: Our study suggests that maternal smoking during pregnancy is associated in a dose-dependent manner with increased risk of RWG in early infancy.

Single oral administration of the novel CXCR4 antagonist, KRH-3955, induces an efficient and long-lasting increase of white blood cell count in normal macaques, and prevents CD4 depletion in SHIV-infected macaques: a preliminary study.

We evaluated the long-term effects of the single oral administration of a new CXCR4 antagonist, KRH-3955, on elevation of white blood cell (WBC), neutrophil and lymphocyte counts in normal cynomolgus monkeys. In the monkeys treated with 0, 2, 20, 200 mg/kg of the compound, WBC, neutrophil and lymphocyte counts increased dramatically at 2 days after treatment. This effect was dose-dependent, and these cell counts remained elevated 15 days after drug treatment. Since neutrophils are the most abundant WBCs in circulation and bone marrow neutrophil exhaustion impairs the response to bacterial infections, it is intriguing to exploit this pharmacological increase of neutrophils as a tool to address its influence on viral infections in vivo. The SHIV infection studies using the SHIV-KS661c/cynomolgus monkey model showed that a single oral administration of KRH-3955 (100 mg/kg) approximately 24 h before virus exposure did not prevent infection, although it did prevent CD4 cell depletion in 3/3 monkeys. Furthermore, single oral administration of the drug 2 weeks before viral exposure rescued CD4 cells in 1/3 monkeys. This prevention of CD4 cell depletion was observed in both blood and lymphoid tissues. These results show that natural course of the SHIV infection is modulated by artificial increase of neutrophils and lymphocytes caused by KRH-3955 in the cynomolgus monkey model.

Double oral administration of emtricitabine/tenofovir prior to virus exposure protects against highly pathogenic simian/human immunodeficiency virus infection in macaques.

In the absence of any effective vaccine against human immunodeficiency virus (HIV), current anti-retroviral drugs may be suitable for pre-exposure prophylaxis (PrEP). Previous large clinical trials showed that PrEP reduced HIV infection in high-risk populations. Emtricitabine/tenofovir (FTC/TDF) may be a suitable agent for PrEP. FTC/TDF PrEP efficacy was evaluated using a highly pathogenic simian/human immunodeficiency virus (SHIV) in a non-human primate model of AIDS, the SHIV-KS661c/cynomolgus monkey model. Double oral administration of FTC/TDF (20/30 mg/kg), at 24 h and a few minutes prior to exposure, completely protected 2/3 monkeys from infection. Interestingly, a single oral administration 2 weeks before viral exposure moderately rescued CD4 cells, although the data did not reach statistical significance. These results are consistent with previous primate studies and with recent clinical data.

Measuring enzymatic HIV-1 susceptibility to two reverse transcriptase inhibitors as a rapid and simple approach to HIV-1 drug-resistance testing.

Simple and cost-effective approaches for HIV drug-resistance testing are highly desirable for managing increasingly expanding HIV-1 infected populations who initiate antiretroviral therapy (ART), particularly in resource-limited settings. Non-nucleoside reverse trancriptase inhibitor (NNRTI)-based regimens with an NRTI backbone containing lamivudine (3TC) or emtricitabine (FTC) are preferred first ART regimens. Failure with these drug combinations typically involves the selection of NNRTI- and/or 3TC/FTC-resistant viruses. Therefore, the availability of simple assays to measure both types of drug resistance is critical. We have developed a high throughput screening test for assessing enzymatic resistance of the HIV-1 RT in plasma to 3TC/FTC and NNRTIs. The test uses the sensitive "Amp-RT" assay with a newly-developed real-time PCR format to screen biochemically for drug resistance in single reactions containing either 3TC-triphosphate (3TC-TP) or nevirapine (NVP). Assay cut-offs were defined based on testing a large panel of subtype B and non-subtype B clinical samples with known genotypic profiles. Enzymatic 3TC resistance correlated well with the presence of M184I/V, and reduced NVP susceptibility was strongly associated with the presence of K103N, Y181C/I, Y188L, and G190A/Q. The sensitivity and specificity for detecting resistance were 97.0% and 96.0% in samples with M184V, and 97.4% and 96.2% for samples with NNRTI mutations, respectively. We further demonstrate the utility of an HIV capture method in plasma by using magnetic beads coated with CD44 antibody that eliminates the need for ultracentifugation. Thus our results support the use of this simple approach for distinguishing WT from NNRTI- or 3TC/FTC-resistant viruses in clinical samples. This enzymatic testing is subtype-independent and can assist in the clinical management of diverse populations particularly in resource-limited settings.

Dominant induction of vaccine antigen-specific cytotoxic T lymphocyte responses after simian immunodeficiency virus challenge.

Cytotoxic T lymphocyte (CTL) responses are crucial for the control of human and simian immunodeficiency virus (HIV and SIV) replication. A promising AIDS vaccine strategy is to induce CTL memory resulting in more effective CTL responses post-viral exposure compared to those in natural HIV infections. We previously developed a CTL-inducing vaccine and showed SIV control in some vaccinated rhesus macaques. These vaccine-based SIV controllers elicited vaccine antigen-specific CTL responses dominantly in the acute phase post-challenge. Here, we examined CTL responses post-challenge in those vaccinated animals that failed to control SIV replication. Unvaccinated rhesus macaques possessing the major histocompatibility complex class I haplotype 90-088-Ij dominantly elicited SIV non-Gag antigen-specific CTL responses after SIV challenge, while those induced with Gag-specific CTL memory by prophylactic vaccination failed to control SIV replication with dominant Gag-specific CTL responses in the acute phase, indicating dominant induction of vaccine antigen-specific CTL responses post-challenge even in non-controllers. Further analysis suggested that prophylactic vaccination results in dominant induction of vaccine antigen-specific CTL responses post-viral exposure but delays SIV non-vaccine antigen-specific CTL responses. These results imply a significant influence of prophylactic vaccination on CTL immunodominance post-viral exposure, providing insights into antigen design in development of a CTL-inducing AIDS vaccine.

Activity of superior interferon α against HIV-1 in severe combined immunodeficient mice reconstituted with human peripheral blood leukocytes.

BACKGROUND: Interferon (IFN) can inhibit human immunodeficiency virus type 1 (HIV-1) replication in vitro and in clinic. However, IFN therapy for HIV infection was limited by its moderate antiviral efficacy and its frequent adverse effects. In the present study we evaluated the anti-HIV efficacy of a novel synthesized superior interferon α (sIFNα). METHODS: We performed in vitro experiments with HIV-1 IIIB infected MT4 cells, and evaluated in vivo anti-HIV efficacy of sIFNα in severe combined immunodeficient (SCID) mice reconstituted with human peripheral blood leukocytes (hu-PBL-SCID mice). RESULTS: We found that the 50% effective concentrations (EC(50)) of sIFNα against the replication of HIV-1 in MT4 cells was 0.06 ng/ml, representing stronger antiviral activity than interferon-α in vitro. In the hu-PBL-SCID mice, a dose-dependent protection pattern was observed: with 0.45 µg and 1.35 µg sIFNα daily treatments, parts of SCID mice were protected from HIV infection, whereas 2.25 µg sIFNα daily treatments resulted in a terminally complete protection. CONCLUSIONS: sIFNα shows good anti-HIV activity both in vitro and in SCID mice, may be a promising anti-HIV agent deserving clinical investigation, especially considering the potential of IFN-α to inhibit HIV replication in patients infected with drug-resistant variants or co-infected with hepatitis C virus (HCV).

Postinfection passive transfer of KD-247 protects against simian/human immunodeficiency virus-induced CD4+ T-cell loss in macaque lymphoid tissue.

BACKGROUND: Preadministration of high-affinity humanized anti-HIV-1 mAb KD-247 by passive transfer provides sterile protection of monkeys from heterologous chimeric simian/human immunodeficiency virus infection. METHODS: Beginning 1 h, 1 day, or 1 week after simian/human immunodeficiency virus-C2/1 challenge (20 50% tissue culture infective dose), mature, male cynomolgus monkeys received multiple passive transfers of KD-247 (45 mg/kg) on a weekly basis for approximately 2 months. Concentrations and viral loads were measured in peripheral blood, and CD4 T-cell counts were examined in both peripheral blood and various lymphoid tissues. RESULTS: Pharmacokinetic examination revealed similar plasma maintenance levels ranging from 200 to 500 microg/ml of KD-247 in the three groups. One of the six monkeys given KD-247 could not maintain these concentrations, and elicitation of anti-KD-247 idiotype antibody was suggested. All monkeys given KD-247 exhibited striking postinfection protection against both CD4 T-cell loss in various lymphoid tissues and atrophic changes in organs compared with control group animals treated with normal human immunoglobulin G. The KD-247-treated groups were also partially protected against plasma viral load elevation in peripheral blood samples, although the complete protection previously reported with preadministration of this mAb was not achieved. CONCLUSION: Postinfection passive transfer of humanized mAb KD-247 with strong neutralizing capacity against challenged virus simian/human immunodeficiency virus-C2/1 protected CD4 T cells in lymphoid organs.

Potential role of natural killer cells in controlling growth and infiltration of AIDS-associated primary effusion lymphoma cells.

Natural killer (NK) cells are an important component of the innate immune response against microbial infections and tumors. Direct involvement of NK cells in tumor growth and infiltration has not yet been demonstrated clearly. Primary effusion lymphoma (PEL) cells were able to produce tumors and ascites very efficiently with infiltration of cells in various organs of T-, B- and NK-cell knock-out NOD/SCID/gammac(null) (NOG) mice within 3 weeks. In contrast, PEL cells formed small tumors at inoculated sites in T- and B-cell knock-out NOD/SCID mice with NK-cells while completely failing to infiltrate into various organs. Immunosupression of NOD/SCID by treatment with an antimurine TM-beta1 antibody, which transiently abrogates NK cell activity in vivo, resulted in enhanced tumorigenicity and organ infiltration in comparison with non-treated NOD/SCID mice. Activated human NK cells inhibited tumor growth and infiltration in NOG mice. Our results suggest that NK cells play an important role in growth and infiltration of PEL cells, and activated NK cells could be a promising immunotherapeutic tool against tumor or virus-infected cells either alone or in combination with conventional therapy. The rapid and efficient engraftment of PEL cells in NOG mice also suggests that this new animal model could provide a unique opportunity to understand and investigate the mechanism of pathogenesis and malignant cell growth.

Cost-effectiveness analysis of antiretroviral drug treatment and HIV-1 vaccination in Thailand.

The prevalence of adult HIV/AIDS in Thailand is declining due to intense prevention strategies, but it still continues to be a critical health problem with a prevalence of 1.5%. Several HIV vaccine candidates for the prevention of HIV infection or progress to AIDS were examined in clinical trials. We evaluated the cost-effectiveness of a vaccination regimen (rBCG prime-rDIs boost) currently in its pre-clinical phase. The cost-effectiveness of three interventions (vaccination, highly active antiretroviral treatment [HAART], and the combination of the two) through an existing vaccination program was assessed in a Markov model. The disability-adjusted life year (DALY) was the main effectiveness measure. In the base case the efficacy of the vaccine for preventing HIV infection was assumed to be 30%. The cost of the vaccine was estimated on the basis of its predicted production capacities in Thailand. The incremental cost-effectiveness ratios of vaccination, HAART, and the combination were about dollar US 75, dollar US 610, and dollar US 267 per DALY averted compared with the do-nothing strategy in the base case. The HAART-only strategy seemed to be less cost-effective than the other options under the current assumptions. Sensitivity analyses indicated that the new HIV infection rate and the vaccine efficacy could affect the results.

Impaired T-cell differentiation in the thymus at the early stages of acute pathogenic chimeric simian-human immunodeficiency virus (SHIV) infection in contrast to less pathogenic SHIV infection.

One of the mechanisms by which HIV infection induces the depletion of CD4+ T cells has been suggested to be impairment of T-cell development in the thymus, although there is no direct evidence that this occurs. To examine this possibility, we compared T-cell maturation in the intrathymic progenitors between macaques infected with an acute pathogenic chimeric simian-human immunodeficiency virus (SHIV), which causes profound and irreversible CD4+ T-cell depletion, and macaques infected with a less pathogenic SHIV, which causes only a transient CD4+ T-cell decline. Within 27 days post-inoculation (dpi), the two virus infections caused similar increases in plasma viral loads and similar decreases in CD4+ T-cell counts. However, in the thymus, the acute pathogenic SHIV resulted in increased thymic involution, atrophy and the depletion of immature T cells including CD4(+)CD8(+) double-positive (DP) cells, whereas the less pathogenic SHIV did not have these effects. Ex vivo differentiation of CD3(-)CD4(-)CD8(-) triple-negative (TN) intrathymic progenitors to DP cells was assessed by a monkey-mouse xenogenic fetal thymus organ culture system. Differentiation was impaired in the TN intrathymic progenitors of the acute pathogenic SHIV-infected monkeys, while differentiation was not impaired in the TN intrathymic progenitors of the less pathogenic SHIV-infected monkeys. These differences suggest that dysfunction of thymic maturation makes an important contribution to the irreversible depletion of circulating CD4+ T cells in vivo.

Anti-V3 humanized antibody KD-247 effectively suppresses ex vivo generation of human immunodeficiency virus type 1 and affords sterile protection of monkeys against a heterologous simian/human immunodeficiency virus infection.

In an accompanying report (Y. Eda, M. Takizawa, T. Murakami, H. Maeda, K. Kimachi, H. Yonemura, S. Koyanagi, K. Shiosaki, H. Higuchi, K. Makizumi, T. Nakashima, K. Osatomi, S. Tokiyoshi, S. Matsushita, N. Yamamoto, and M. Honda, J. Virol. 80:5552-5562, 2006), we discuss our production of a high-affinity humanized monoclonal antibody, KD-247, by sequential immunization with V3 peptides derived from human immunodeficiency virus type 1 (HIV-1) clade B primary isolates. Epitope mapping revealed that KD-247 recognized the Pro-Gly-Arg V3 tip sequence conserved in HIV-1 clade B isolates. In this study, we further demonstrate that in vitro, KD-247 efficiently neutralizes CXCR4- and CCR5-tropic primary HIV-1 clade B and clade B' with matching neutralization sequence motifs but does not neutralize sequence-mismatched clade B and clade E isolates. Monkeys were provided sterile protection against heterologous simian/human immunodeficiency virus challenge by the passive transfer of a single high dose (45 mg per kg of body weight) of KD-247 and afforded partial protection by lower antibody doses (30 and 15 mg per kg). Protective neutralization endpoint titers in plasma at the time of virus challenge were 1:160 in animals passively transferred with a high dose of the antibody. The antiviral efficacy of the antibody was further confirmed by its suppression of the ex vivo generation of primary HIV-1 quasispecies in peripheral blood mononuclear cell cultures from HIV-infected individuals. Therefore, KD-247 promises to be a valuable tool not only as a passive immunization antibody for the prevention of HIV infection but also as an immunotherapy for the suppression of HIV in phenotype-matched HIV-infected individuals.

Rapid dissemination of a pathogenic simian/human immunodeficiency virus to systemic organs and active replication in lymphoid tissues following intrarectal infection.

A better understanding of virological events during the early phase of human immunodeficiency virus 1 (HIV-1) infection is important for development of effective antiviral vaccines. In this study, by using quantitative PCR and an infectious plaque assay, virus distribution and replication were examined in various internal organs of rhesus macaques for almost 1 month after intrarectal inoculation of a pathogenic simian immunodeficiency virus/HIV chimeric virus (SHIV-C2/1-KS661c). At 3 days post-inoculation (p.i.), proviral DNA was detected in the rectum, thymus and axillary lymph node. In lymphoid tissues, infectious virus was first detected at 6 days p.i. and a high level of proviral DNA and infectious virus were both detected at 13 days p.i. By 27 days p.i., levels of infectious virus decreased dramatically, although proviral DNA load remained unaltered. In the intestinal tract, levels of infectious virus detected were much lower than in lymphoid tissues, whereas proviral DNA was detected at the same level as in lymphoid tissues throughout the infection. In the thymus and jejunum, CD4CD8 double-positive T cells were depleted earlier than CD4 single-positive cells. These results show that the virus spread quickly to systemic tissues after mucosal transmission. Thereafter, infectious virus was actively produced in the lymphoid tissues, but levels decreased significantly after the peak of viraemia. In contrast, in the intestinal tract, infectious virus was produced at low levels from the beginning of infection. Moreover, virus pathogenesis differed in CD4 single-positive and CD4CD8 double-positive T cells.

Induction of positive cellular and humoral immune responses by a prime-boost vaccine encoded with simian immunodeficiency virus gag/pol.

It is believed likely that immune responses are responsible for controlling viral load and infection. In this study, when macaques were primed with plasmid DNA encoding SIV gag and pol genes (SIVgag/pol DNA) and then boosted with replication-deficient vaccinia virus DIs recombinant expressing the same genes (rDIsSIVgag/pol), this prime-boost regimen generated higher levels of Gag-specific CD4+ and CD8+ T cell responses than did either SIVgag/pol DNA or rDIsSIVgag/pol alone. When the macaques were i.v. challenged with pathogenic simian/HIV, the prime-boost group maintained high CD4+ T cell counts and reduced plasma viral loads up to 30 wk after viral challenge, whereas the rDIsSIVgag/pol group showed only a partial attenuation of the viral infection, and the group immunized with SIVgag/pol DNA alone showed none at all. The protection levels were better correlated with the levels of virus-specific T cell responses than the levels of neutralization Ab responses. These results demonstrate that a vaccine regimen that primes with DNA and then boosts with a replication-defective vaccinia virus DIs generates anti-SIV immunity, suggesting that it will be a promising vaccine regimen for HIV-1 vaccine development.

Effects of immunization with CCR5-based cycloimmunogen on simian/HIVSF162P3 challenge.

A synthetic cycloimmunogen targeting the HIV-1 coreceptor CCR5 was evaluated for its capacity to induce CCR5-specific Abs with anti-HIV-1 activity in cynomolgus macaques. The cyclic closed-chain dodecapeptide (cDDR5) mimicking the conformation-specific domain of human CCR5 was chemically prepared, in which the Gly-Glu dipeptide links the amino and carboxy termini of the decapeptidyl linear chain (Arg168 to Thr177) derived from the undecapeptidyl arch (Arg168 to Cys178) of extracellular loop-2 in CCR5. The immunization of cynomolgus macaques with the cDDR5-conjugated multiple-Ag peptide (cDDR5-MAP) induced anti-cDDR5 serum production for approximately 15 wk after the third immunization. The antisera raised against cDDR5-MAP reacted with both human and macaque CCR5s, and potently suppressed infection by the R5 HIV-1 laboratory isolate (HIV JRFL), R5 HIV-1 primary isolates (clade A:HIV 93RW004 and clade C:HIV MJ4), and a pathogenic simian/HIV (SHIV SF162P3) bulk isolate in vitro. To examine the prophylactic efficacy of anti-CCR5 serum Ab for acute HIV-1 infection, cynomolgus macaques were challenged with SHIV SF162P3. The cDDR5-MAP immunization attenuated the acute phase of SHIV SF162P3 replication. The geometric mean plasma viral load in the vaccinated macaques was 217.10 times lower than that of the control macaques at 1 wk postchallenge. Taken together, these results suggest that cDDR5-MAP immunization is an effective prophylactic vaccine strategy that suppresses and delays viral propagation during the initial HIV-1 transmission for the containment of HIV-1 replication subsequent to infection.

Properties of anti-gp41 core structure antibodies, which compete with sera of HIV-1-infected patients.

To determine the correlation between the immunoreaction against the core structure of human immunodeficiency virus type (HIV-1) transmembrane protein gp41 epitopes and the disease progression, it is essential to evaluate the anti-core structure antibody epitopes and the humoral immunity against the epitopes. For this purpose we evaluated monoclonal antibodies (mAbs) against the gp41 core structure such as mAbs 50.69, 98.6 and T26, by Western blotting (WB) and flow cytometry. WB showed mAbs 50.69 and 98.6 bound to both monomeric and oligomeric gp41, and mAb T26 exclusively bound to oligomeric gp41. We evaluated the sera from Pneumocystis pneumonia patients (PCP; n=7) and long-term survivors (LTS; n=7). Competition assay with sera and mAbs for binding to H9 cells infected with HIV-1 IIIB virus was done using flow cytometry. The results revealed that PCP sera as well as LTS sera inhibited the binding of all the three mAbs, and the PCP sera inhibited mAb T26 binding more efficiently than LTS. Therefore, PCP patients retain competing immunity to antibodies against not only the shared epitopes of the core structure (binding sites of mAbs 50.69 and 98.6) but also against oligomeric gp41 specific epitope (binding site of mAb T26).

Vaccination of rhesus macaques with recombinant Mycobacterium bovis bacillus Calmette-Guérin Env V3 elicits neutralizing antibody-mediated protection against simian-human immunodeficiency virus with a homologous but not a heterologous V3 motif.

Although the correlates of vaccine-induced protection against human immunodeficiency virus type 1 (HIV-1) are not fully known, it is presumed that neutralizing antibodies (NAb) play a role in controlling virus infection. In this study, we examined immune responses elicited in rhesus macaques following vaccination with recombinant Mycobacterium bovis bacillus Calmette-Guérin expressing an HIV-1 Env V3 antigen (rBCG Env V3). We also determined the effect of vaccination on protection against challenge with either a simian-human immunodeficiency virus (SHIV-MN) or a highly pathogenic SHIV strain (SHIV-89.6PD). Immunization with rBCG Env V3 elicited significant levels of NAb for the 24 weeks tested that were predominantly HIV-1 type specific. Sera from the immunized macaques neutralized primary HIV-1 isolates in vitro, including HIV-1BZ167/X4, HIV-1SF2/X4, HIV-1CI2/X4, and, to a lesser extent, HIV-1MNp/X4, all of which contain a V3 sequence homologous to that of rBCG Env V3. In contrast, neutralization was not observed against HIV-1SF33/X4, which has a heterologous V3 sequence, nor was it found against primary HIV-1 R5 isolates from either clade A or B. Furthermore, the viral load in the vaccinated macaques was significantly reduced following low-dose challenge with SHIV-MN, and early plasma viremia was markedly decreased after high-dose SHIV-MN challenge. In contrast, replication of pathogenic SHIV-89.6PD was not affected by vaccination in any of the macaques. Thus, we have shown that immunization with an rBCG Env V3 vaccine elicits a strong, type-specific V3 NAb response in rhesus macaques. While this response was not sufficient to provide protection against a pathogenic SHIV challenge, it was able to significantly reduce the viral load in macaques following challenge with a nonpathogenic SHIV. These observations suggest that rBCG vectors have the potential to deliver an appropriate virus immunogen for desirable immune elicitations.

Higher levels of IL-18 circulate during primary infection of monkeys with a pathogenic SHIV than with a nonpathogenic SHIV.

We have monitored kinetics of peripheral blood Interleukin (IL)-18 level, viral RNA load, and CD4(+) T cell counts in cynomolgus and rhesus macaques following infections of various simian/human immunodeficiency viruses (SHIVs) causing differential pathogenicity. Infections of cynomolgus and rhesus macaques with pathogenic SHIVs-C2/1 and -89.6PD, respectively, induced high levels of plasma IL-18 (0.1-1 ng/ml) and enhanced apoptosis of peripheral blood T cells during primary viremia, along with a rapid decline of CD4(+) T cells and a high level of set point viral load after primary viremia (six of six cases). In contrast, infections of cynomolgus macaques with nonpathogenic SHIVs-TH09V3 and -MD14 did not cause such IL-18 elevation, showing no decline of CD4(+) T cells and no or low viral set point level following primary viremia (three of three cases). Thus, the elevation of circulating IL-18 level during primary viral infection can be a good indicator of an active pathogenic viral infection. However, the role of increased IL-18 remains to be elucidated and needs further investigation.

Assessment of oral transmission using cell-free human immunodeficiency virus-1 in mice reconstituted with human peripheral blood leucocyte.

Oral-genital contact is one of the risk factors for the transmission of human immunodeficiency virus (HIV) in adults. In recent reports, oral exposure to simian immunodeficiency virus (SIV) was found to have important implications for the achievement of mucosal transmission of HIV in a rhesus monkey animal model. In the present study, we aimed first to establish a small animal model which did not develop tonsils suitable for HIV oral mucosa transmission, using non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice and NOD/SCID B2m(null) mice grafted with human peripheral blood leucocytes (hu-PBL) and stimulated with interleukin (IL)-4, and second to investigate whether oral exposure to cell-free R5 and X4 HIV-1 could lead to oral transmission of HIV through intact or traumatized mucosal tissues in humanized mice. Both low and high concentrations of cell-free R5 and X4 viruses failed to cause oral transmission with or without trauma in hu-PBL-NOD/SCID and NOD/SCID Beta2m(null) mice, which presented a number of CD4+ cells in gingival tissues and oral cavities with or without tissue injury. The present results show that IL-4-administrated NOD/SCID B2m(null) mice are useful as a small-humanized model for the study of HIV oral infection, which may help to define the window of opportunity for oral transmission by the HIV virus in animal model experiments.

Combined intrarectal/intradermal inoculation of recombinant Mycobacterium bovis bacillus Calmette-Guérin (BCG) induces enhanced immune responses against the inserted HIV-1 V3 antigen.

The development of a successful recombinant Mycobacterium bovis bacillus Calmette-Guérin (rBCG) vector-based vaccine for human immunodeficiency virus type 1 (HIV-1) requires the induction of high levels of HIV-1-specific immunity while at the same time maintaining immunity to tuberculosis. To examine a combined vaccination strategy for enhancement of immune responses specific for HIV-1, guinea pigs were inoculated with either a single or combination intradermal (i.d.), intrarectal (i.r.) and intranasal (i.n.) administration of rBCG-pSOV3J1 which secretes a chimeric protein of HIV-1 V3J1 peptide and alpha-antigen. Significant level of delayed-type hypersensitivity to both V3J1 peptide and tuberculin was induced in guinea pigs inoculated with human doses of rBCG-pSOV3J1 by a combination of intrarectal and intradermal routes. Guinea pigs inoculated by combined routes also had significantly higher titers of HIV-1-specific serum IgG and IgA compared with those animals immunized only intrarectally, which led to the enhanced neutralization activity against HIV-1(MN). In addition, the induction of high levels of IFNgamma and interleukin-2 (IL-2) mRNA in PBMC, splenocytes, and intraepithelial lymphocytes from the immunized animals was detected until at least 110 weeks post-inoculation. These results suggest that enhanced immune responses specific for HIV-1 are efficiently induced by combined intrarectal and intradermal immunization with rBCG-HIV, and antigen-specific Th1-type memory cells are maintained for more than 2 years in the immunized animals. Thus, inoculation with rBCG-HIV by combined routes represents an effective vaccination strategy to elicit high levels of HIV-1-specific immune responses.