Reduction of simian-human immunodeficiency virus 89.6P viremia in rhesus monkeys by recombinant modified vaccinia virus Ankara vaccination

Since cytotoxic T lymphocytes (CTLs) are critical for controlling human immunodeficiency virus type 1 (HIV-1) replication in infected individuals, candidate HIV-1 vaccines should elicit virus-specific CTL responses. In this report, we study the immune responses elicited in rhesus monkeys by a recombinant poxvirus vaccine and the degree of protection afforded against a pathogenic simian-human immunodeficiency virus SHIV-89.6P challenge. Immunization with recombinant modified vaccinia virus Ankara (MVA) vectors expressing SIVmac239 gag-pol and HIV-1 89.6 env elicited potent Gag-specific CTL responses but no detectable SHIV-specific neutralizing antibody (NAb) responses. Following intravenous SHIV-89.6P challenge, sham-vaccinated monkeys developed low-frequency CTL responses, low-titer NAb responses, rapid loss of CD4+ T lymphocytes, high-setpoint viral RNA levels, and significant clinical disease progression and death in half of the animals by day 168 postchallenge. In contrast, the recombinant MVA-vaccinated monkeys demonstrated high-frequency secondary CTL responses, high-titer secondary SHIV-89.6-specific NAb responses, rapid emergence of SHIV-89.6P-specific NAb responses, partial preservation of CD4+ T lymphocytes, reduced setpoint viral RNA levels, and no evidence of clinical disease or mortality by day 168 postchallenge. There was a statistically significant correlation between levels of vaccine-elicited CTL responses prior to challenge and the control of viremia following challenge. These results demonstrate that immune responses elicited by live recombinant vectors, although unable to provide sterilizing immunity, can control viremia and prevent disease progression following a highly pathogenic AIDS virus challenge.     

Effect of humoral immune responses on controlling viremia during primary infection of rhesus monkeys with simian immunodeficiency virus

Cellular immune responses mediated by CD8+ lymphocytes exert efficient control of virus replication during primary simian immunodeficiency virus (SIV) infection. However, the role that antibodies may play in the early control of virus replication remains unclear. To evaluate how antibody responses may affect virus replication during primary SIVmac infection, we depleted rhesus monkeys of B cells with anti-CD20 antibody. In normal rhesus monkeys immunized with tetanus toxoid, anti-CD20 treatment and resulting depletion of B cells inhibited the generation of antitetanus antibodies, while tetanus-specific T-cell responses were preserved. During the first 4 weeks after inoculation with SIVmac251, development of SIV-specific neutralizing antibody was delayed, and titers were significantly lower in B-cell-depleted monkeys than control-antibody-treated monkeys. Despite the lower neutralizing antibody titers, the levels of plasma SIV RNA and the linear slope of the decline seen in B-cell-depleted monkeys did not differ from that observed in monkeys treated with control antibody. However, beginning at day 28 after SIV infection, the B-cell-depleted monkeys showed a significant inverse correlation between neutralizing antibody titers and plasma virus level. These results suggest that the rapid decline of peak viremia that typically occurs during the first 3 weeks of infection was not significantly affected by SIV-specific antibodies. However, the inverse correlation between neutralizing antibodies and plasma virus level during the postacute phases of infection suggests that humoral immune responses may contribute to the control of SIV replication.     

A tetravalent dengue vaccine based on a complex adenovirus vector provides significant protection in rhesus monkeys against all four serotypes of dengue virus

Nearly a third of the human population is at risk of infection with the four serotypes of dengue viruses, and it is estimated that more than 100 million infections occur each year. A licensed vaccine for dengue viruses has become a global health priority. A major challenge to developing a dengue vaccine is the necessity to produce fairly uniform protective immune responses to all four dengue virus serotypes. We have developed two bivalent dengue virus vaccines, using a complex adenovirus vector, by incorporating the genes expressing premembrane (prM) and envelope (E) proteins of dengue virus types 1 and 2 (dengue-1 and -2, respectively) (CAdVax-Den12) or dengue-3 and -4 (CAdVax-Den34). Rhesus macaques were vaccinated by intramuscular inoculation of a tetravalent dengue vaccine formulated by combining the two bivalent vaccine constructs. Vaccinated animals produced high-titer antibodies that neutralized all four serotypes of dengue viruses in vitro. The ability of the vaccine to induce rapid, as well as sustained, protective immune responses was examined with two separate live-virus challenges administered at 4 and 24 weeks after the final vaccination. For both of these virus challenge studies, significant protection from viremia was demonstrated for all four dengue virus serotypes in vaccinated animals. Viremia from dengue-1 and dengue-3 challenges was completely blocked, whereas viremia from dengue-2 and dengue-4 was significantly reduced, as well as delayed, compared to that of control-vaccinated animals. These results demonstrate that the tetravalent dengue vaccine formulation provides significant protection in rhesus macaques against challenge with all four dengue virus serotypes.     

Glutathione and transferrin in rhesus monkeys1

Transferrin types and glutathione (GSH) levels were determined for 38 rhesus monkeys (Macaca mulatto). Eight transferrin alleles were found: their gene frequencies were: Tf^A 0.04, Tf^c 0.50, Tf^D 0.03, Tf^D 0.09, TF 0.11, TF 0.05, Tf^G 0.11, and Tf ^H' 0.08. The 16 phenotypes found and the number of each was: Tf AC 3, Tf CC 9, Tf CD‘ 2, Tf CD 2, Tf CF 5, Tf CF 3, Tf CH’ 3, Tf CG 2, Tf D'D‘ 1, Tf D'F 1, Tf D'G 1, TfD'H’ l, TfFG2, TfF'G ‘l.TfCG l.and Tf H'H’ 1. Mean GSH level was 310.6 mg/100 ml red cells. The Tf CC group had significantly lower GSH levels than Tf^c heterozygotes or non-Tf^c homozygotes. The Tf^c heterozygotes had significantly lower GSH levels than non-Tf^c homozygotes. Tf^c, Tf^D' and TF alleles were associated with lower GSH levels. Glutathione levels were higher in older animals and were higher in September than in December. Sex differences in glutathione levels were not found.     

Characterization of antibody responses to combinations of a dengue virus type 2 DNA vaccine and two dengue virus type 2 protein vaccines in rhesus macaques

We evaluated three nonreplicating dengue virus type 2 (DENV-2) vaccines: (i) a DNA vaccine containing the prM-E gene region (D), (ii) a recombinant subunit protein vaccine containing the B domain (i.e., domain III) of the E protein as a fusion with the Escherichia coli maltose-binding protein (R), and (iii) a purified inactivated virus vaccine (P). Groups of four rhesus macaques each were primed once and boosted twice using seven different vaccination regimens. After primary vaccination, enzyme-linked immunosorbent assay (ELISA) antibody levels increased most rapidly for groups inoculated with the P and DP combination, and by 1 month after the second boost, ELISA titers were similar for all groups. The highest plaque reduction neutralization test (PRNT) titers were seen in those groups that received the DR/DR/DR combination (geometric mean titer [GMT], 510), the P/P/P vaccine (GMT, 345), the DP/DP/DP combination (GMT, 287), and the R/R/R vaccine (GMT, 200). The next highest titers were seen in animals that received the D/R/R vaccine (GMT, 186) and the D/P/P vaccine (GMT, 163). Animals that received the D/D/D vaccine had the lowest neutralizing antibody titer (GMT, 49). Both ELISA and PRNT titers declined at variable rates. The only significant protection from viremia was observed in the P-vaccinated animals (mean of 0.5 days), which also showed the highest antibody concentration, including antibodies to NS1, and highest antibody avidity at the time of challenge.     

Kinetics of viremia and NS1 antigenemia are shaped by immune status and virus serotype in adults with dengue

Background

Dengue is a major public health problem in tropical and subtropical countries. Exploring the relationships between virological features of infection with patient immune status and outcome may help to identify predictors of disease severity and enable rational therapeutic strategies.

Methods

Clinical features, antibody responses and virological markers were characterized in Vietnamese adults participating in a randomised controlled treatment trial of chloroquine.

Results

Of the 248 patients with laboratory-confirmed dengue and defined serological and clinical classifications 29 (11.7%) had primary DF, 150 (60.5%) had secondary DF, 4 (1.6%) had primary DHF and 65 (26.2%) had secondary DHF. DENV-1 was the commonest serotype (57.3%), then DENV-2 (20.6%), DENV-3 (15.7%) and DENV-4 (2.8%). DHF was associated with secondary infection (Odds ratio = 3.13, 95% CI 1.04–12.75). DENV-1 infections resulted in significantly higher viremia levels than DENV-2 infections. Early viremia levels were higher in DENV-1 patients with DHF than with DF, even if the peak viremia level was often not observed because it occurred prior to enrolment. Peak viremias were significantly less often observed during secondary infections than primary for all disease severity grades (P = 0.001). The clearance of DENV viremia and NS1 antigenemia occurs earlier and faster in patients with secondary dengue (P<0.0001). The maximum daily rate of viremia clearance was significantly higher in patients with secondary infections than primary (P<0.00001).

Conclusions

Collectively, our findings suggest that the early magnitude of viremia is positively associated with disease severity. The clearance of DENV is associated with immune status, and there are serotype dependent differences in infection kinetics. These findings are relevant for the rational design of randomized controlled trials of therapeutic interventions, especially antivirals.     

Genome 3'-end repair in dengue virus type 2

Genomes of RNA viruses encounter a continual threat from host cellular ribonucleases. Therefore, viruses have evolved mechanisms to protect the integrity of their genomes. To study the mechanism of 3′-end repair in dengue virus-2 in mammalian cells, a series of 3′-end deletions in the genome were evaluated for virus replication by detection of viral antigen NS1 and by sequence analysis. Limited deletions did not cause any delay in the detection of NS1 within 5 d. However, deletions of 7–10 nucleotides caused a delay of 9 d in the detection of NS1. Sequence analysis of RNAs from recovered viruses showed that at early times, virus progenies evolved through RNA molecules of heterogeneous lengths and nucleotide sequences at the 3′ end, suggesting a possible role for terminal nucleotidyl transferase activity of the viral polymerase (NS5). However, this diversity gradually diminished and consensus sequences emerged. Template activities of 3′-end mutants in the synthesis of negative-strand RNA in vitro by purified NS5 correlate well with the abilities of mutant RNAs to repair and produce virus progenies. Using the Mfold program for RNA structure prediction, we show that if the 3′ stem–loop (3′ SL) structure was abrogated by mutations, viruses eventually restored the 3′ SL structure. Taken together, these results favor a two-step repair process: non-template-based nucleotide addition followed by evolutionary selection of 3′-end sequences based on the best-fit RNA structure that can support viral replication.     

Recombinant, live-attenuated tetravalent dengue virus vaccine formulations induce a balanced, broad, and protective neutralizing antibody response against each of the four serotypes in rhesus monkeys

Three tetravalent vaccine (TV) formulations of previously described monovalent dengue (DEN) virus vaccine candidates were compared to a tetravalent formulation of wild-type DEN viruses (T-wt) for replication in SCID mice transplanted with human liver cells (SCID-HuH-7) or for replication and immunogenicity in rhesus monkeys. TV-1 consists of recombinant DEN1, -2, -3, and -4, each with a 30-nucleotide deletion in the 3' untranslated region (Delta30). TV-2 consists of rDEN1Delta30, rDEN4Delta30, and two antigenic chimeric viruses, rDEN2/4Delta30 and rDEN3/4Delta30, both also bearing the Delta30 mutation. TV-3 consists of rDEN1Delta30, rDEN2Delta30, rDEN4Delta30, and a 10-fold higher dose of rDEN3/4Delta30. TV-1 and TV-2 were attenuated in SCID-HuH-7 mice with minimal interference in replication among the virus components. TV-1, -2, and -3 were attenuated in rhesus monkeys as measured by duration and peak of viremia. Each monkey immunized with TV-1 and TV-3 seroconverted to the four DEN components by day 28 with neutralization titers ranging from 1:52 to 1:273 and 1:59 to 1:144 for TV-1 and TV-3, respectively. TV-2 induced low antibody titers to DEN2 and DEN3, but a booster immunization after 4 months increased the neutralizing antibody titers to greater than 1:100 against each serotype and elicited broad neutralizing activity against 19 of 20 DEN subtypes. A single dose of TV-2 induced protection against wild-type DEN1, DEN3, and DEN4 challenge, but not DEN2. However, two doses of TV-2 or TV-3 induced protection against DEN2 challenge. Two tetravalent formulations, TV-2 and TV-3, possess properties of a successful DEN vaccine and can be considered for evaluation in clinical trials.     

Unusual dengue virus 3 epidemic in Nicaragua, 2009

The four dengue virus serotypes (DENV1-4) cause the most prevalent mosquito-borne viral disease affecting humans worldwide. In 2009, Nicaragua experienced the largest dengue epidemic in over a decade, marked by unusual clinical presentation, as observed in two prospective studies of pediatric dengue in Managua. From August 2009-January 2010, 212 dengue cases were confirmed among 396 study participants at the National Pediatric Reference Hospital. In our parallel community-based cohort study, 170 dengue cases were recorded in 2009-10, compared to 13-65 cases in 2004-9. In both studies, significantly more patients experienced "compensated shock" (poor capillary refill plus cold extremities, tachycardia, tachypnea, and/or weak pulse) in 2009-10 than in previous years (42.5% [90/212] vs. 24.7% [82/332] in the hospital study (p<0.001) and 17% [29/170] vs. 2.2% [4/181] in the cohort study (p<0.001). Signs of poor peripheral perfusion presented significantly earlier (1-2 days) in 2009-10 than in previous years according to Kaplan-Meier survival analysis. In the hospital study, 19.8% of subjects were transferred to intensive care, compared to 7.1% in previous years - similar to the cohort study. DENV-3 predominated in 2008-9, 2009-10, and 2010-11, and full-length sequencing revealed no major genetic changes from 2008-9 to 2010-11. In 2008-9 and 2010-11, typical dengue was observed; only in 2009-10 was unusual presentation noted. Multivariate analysis revealed only "2009-10" as a significant risk factor for Dengue Fever with Compensated Shock. Interestingly, circulation of pandemic influenza A-H1N1 2009 in Managua was shifted such that it overlapped with the dengue epidemic. We hypothesize that prior influenza A H1N1 2009 infection may have modulated subsequent DENV infection, and initial results of an ongoing study suggest increased risk of shock among children with anti-H1N1-2009 antibodies. This study demonstrates that parameters other than serotype, viral genomic sequence, immune status, and sequence of serotypes can play a role in modulating dengue disease outcome.     

Chimeric dengue type 2 (vaccine strain PDK-53)/dengue type 1 virus as a potential candidate dengue type 1 virus vaccine

We constructed chimeric dengue type 2/type 1 (DEN-2/DEN-1) viruses containing the nonstructural genes of DEN-2 16681 virus or its vaccine derivative, strain PDK-53, and the structural genes (encoding capsid protein, premembrane protein, and envelope glycoprotein) of DEN-1 16007 virus or its vaccine derivative, strain PDK-13. We previously reported that attenuation markers of DEN-2 PDK-53 virus were encoded by genetic loci located outside the structural gene region of the PDK-53 virus genome. Chimeric viruses containing the nonstructural genes of DEN-2 PDK-53 virus and the structural genes of the parental DEN-1 16007 virus retained the attenuation markers of small plaque size and temperature sensitivity in LLC-MK(2) cells, less efficient replication in C6/36 cells, and attenuation for mice. These chimeric viruses elicited higher mouse neutralizing antibody titers against DEN-1 virus than did the candidate DEN-1 PDK-13 vaccine virus or chimeric DEN-2/DEN-1 viruses containing the structural genes of the PDK-13 virus. Mutations in the envelope protein of DEN-1 PDK-13 virus affected in vitro phenotype and immunogenicity in mice. The current PDK-13 vaccine is the least efficient of the four Mahidol candidate DEN virus vaccines in human trials. The chimeric DEN-2/DEN-1 virus might be a potential DEN-1 virus vaccine candidate. This study indicated that the infectious clones derived from the candidate DEN-2 PDK-53 vaccine are promising attenuated vectors for development of chimeric flavivirus vaccines.     

Epstein-Barr virus types 1 and 2 differ in their EBNA-3A, EBNA-3B, and EBNA-3C genes.

The two Epstein-Barr virus (EBV) types, EBV-1 and EBV-2, are known to differ in their EBNA-2 genes, which are 64 and 53% identical in their nucleotide and predicted amino acid sequences, respectively. Restriction endonuclease maps and serologic analyses detect few other differences between EBV-1 and EBV-2 except in the EBNA-3 gene family. We determined the DNA sequence of the AG876 EBV-2 EBNA-3 coding region and have compared it with known B95-8 EBV-1 EBNA-3 sequences to delineate the extent of divergence between EBV-1 and EBV-2 isolates in their EBNA-3 genes. The B95-8 and AG876 EBV isolates had nucleotide and amino acid identity levels of 90 and 84%, 88 and 80%, and 81 and 72% for the EBNA-3A, -3B, and -3C genes, respectively. In contrast, nucleotide sequence identity in the noncoding DNA adjacent to the B95-8 and AG876 EBNA-3 open reading frames was 96%. We used the polymerase chain reaction to demonstrate that five additional EBV-1 isolates and six additional EBV-2 isolates have the type-specific differences in their EBNA-3 genes predicted from the B95-8 or AG876 sequences. Thus, EBV-1 and EBV-2 are two distinct wild-type EBV strains that have significantly diverged at four genetic loci and have maintained type-characteristic differences at each locus. The delineation of these sequence differences between EBV-1 and EBV-2 is essential to ongoing molecular dissection of the biologic properties of EBV and of the human immune response to EBV infection. The application of these data to the delineation of epitopes recognized in the EBV-immune T-cell response is also discussed.     

Self-agency in rhesus monkeys

Rhesus monkeys (Macaca mulatta) have shown the ability to monitor their own mental states, but fail the mirror self-recognition test. In humans, the sense of self-agency is closely related to self-awareness, and results from monitoring the relationship between intentional, sensorimotor and perceptual information. Humans and rhesus monkeys were trained to move a computer icon with a joystick while a distractor icon partially matched their movements. Both humans and monkeys were able to monitor and identify the icon they were controlling, suggesting they have some understanding of self-agency.     

Tactile-spatial and cross-modal attention effects in the primary somatosensory cortical areas 3b and 1-2 of rhesus monkeys

Neuronal responses in somatosensory cortical areas 3b and 1-2 (S1) were recorded during an attention task involving cue directed selection of one of three simultaneous stimuli: dual sinewave shaped vibrotactile stimuli applied to mirror sites on both hands or a similarly timed auditory tone. The cued stimulus occurred with one of two equally probable patterns: a constant amplitude vibration or the latter with a superimposed brief sinewave amplitude pulse midway during stimulation. Uncued stimuli always contained amplitude pulses. Two monkeys signaled the absence or presence of an amplitude pulse by appropriately moving a foot pedal up or down. Cues initiated trials by marking the location where the monkey had to discriminate the stimulus pattern. Cue location and stimulus pattern varied randomly per trial. Approximately 50% of cells (44/77 in 3b and 39/77 in 1-2) had significantly different firing rates to stimulation cued to the contralateral hand relative to spatially cuing the ipsilateral hand or cross-modally the auditory stimulus. Relatively suppressed firing rates during times prior to the epoch containing amplitude pulses improved signal-to-noise ratios for responses to amplitude pulses. Instances of significant enhanced activity during and after intervals with amplitude pulses were rare and relative to suppressed activity when cues directed attention to the ipsilateral hand or auditory stimulus. The present findings suggest that attention influences even the earliest stage somatosensory cortical processing. Findings were more modest in S1 than those previously seen in S2 (Burton et al., Somatosens Mot Res 14: 237-267, 1997), which supports the concept of multistage attention processes for touch.     

Endocardiosis in rhesus monkeys

Endocardiosis was diagnosed as an incidental finding in two rhesus monkeys. The gross and histologic appearance of the lesions was described, and the similarity of this lesion to lesions of endocardiosis as found in dogs and man was discussed.The animals used in this study were handled in accordance with the Guide for Laboratory Animal Facilities and Care established by the National Academy of Science, National Research Council.     

Epidemiology of parainfluenza virus types 1, 2 and 3 infections based on virus isolation between 2002 and 2011 in Yamagata,Japan

To clarify the epidemiology of viral acute respiratory infections (ARIs), 305 human parainfluenza virus types 1 (HPIV1), 154 HPIV2 and 574 HPIV3 strains were isolated from 16,962 nasopharyngeal swabs obtained between 2002 and 2011 at pediatric clinics in Yamagata, Japan. The total isolation frequency for HPIV1–3 was 6.1%. Unlike HPIV1 infections, HPIV3 showed clear seasonality with yearly outbreaks in the spring–summer season. HPIV2 tended to appear biannually in autumn–winter. Although no reliable techniques for the laboratory diagnosis of these infections have been established, the present results suggest that HPIV1–3 are an important causative agent of ARIs in children.     

Pharmacokinetics of recombinant human interleukin-3 in rhesus monkeys

Concentration-time profiles of ~(125)I-labeled recombinant human interleukin-3 (125IrhIL-3) were de-termined by reverse phase high performance liquid chromatography (RHPLC) after intravenous and subcutaneous ad-ministration of the drug in 16 rhesus monkeys. The initial and terminal T1/2 in plasma after intravenous of 30 μg/kg were (0. 15 ±0.13) and (2. 21 ± 0. 59) h, respectively. Terminal half-lives after 30, 90 and 180μg/kg subcutaneous (s.c.) injections were 2. 0-3.8 h. Area under concentration-time curves (AUC) following s. c. were roughly in-creased with dose, while CL5 were similar among different dosages. The absorption rates were dependent on concentra-tion at injected site. Bioavailability was about 0.7 after s.c. Rapid biodegradation was found in plasma. Distribution profiles of total radioactivity were as follows: the highest level was found in urinary system; levels in bile-enteric sys-tem, lymph nodes, bone marrow and spleen were near to that in plasma, and level in brain was the lowest