Molecular characteristics of rhesus macaque interleukin-22: cloning, in vitro expression and biological activities.

Interleukin-22 (IL-22) is a potential therapeutic agent for diseases driven by epithelial injury. To characterize the IL-22 expressed by rhesus macaques, animals that are irreplaceable for human disease research, rhesus macaque IL-22 (rhIL-22) was cloned and expressed, and its biological activity and in vivo distribution were examined. It was found that the rhIL-22 gene consists of five introns and six exons, including a short non-coding exon starting 22 bp downstream of a putative TATA box. The amino acid sequence of rhIL-22 showed 95·5% identity to that of humans, and it shared two conserved disulphide bonds, three N-glycosylation sites and all the critical residues for binding to IL-22R1. High levels of IL-22 mRNA were observed in the liver, pancreas, lymphoid tissues and especially in the outer-body barriers such as the intestinal tract of rhesus macaques. Functionally, purified rhIL-22 has a similar but a little earlier effect on signal transducer and activator of transcription 3 phosphorylation at Tyr705 compared with that of commercial human IL-22. The expression of the antibacterial proteins ß-defensin-2, S100A8, S100A9, RegIIIα and Muc1 by HT-29 cells was largely upregulated after stimulation with rhIL-22. Recombinant rhIL-22 could also significantly promote the proliferation of human intestinal epithelial cells without affecting cell apoptosis. These data indicate that rhesus macaque IL-22 is highly similar to that of humans in both structure and function, and tests of therapeutic effects of human IL-22 on human diseases in rhesus macaques are warranted.

Associated changes in the transcription levels of IL-17A and tight junction-associated genes in the duodenal mucosa of rhesus macaques repeatedly exposed to simian/human immunodeficiency virus.

BACKGROUND: Mucosal barrier dysfunction might play a key role in HIV/AIDS, yet the early effects of HIV-1 on intestinal mucosal barrier, especially tight junctions (TJ) have not been well addressed. AIMS: To investigate the effects of acute HIV-1 infection on the expression of intestinal IL-17A and TJ-associated genes using an NHP-AIDS model. METHODS: TaqMan probe real-time RT-PCR methods were established and claudin-1, claudin-3, occludin and zonula occluden-1 (ZO-1) mRNA levels in the duodenal biopsies of rhesus macaques collected before and after rectal exposures to SHIV-SF162P4 were examined and compared with that of IL-17A, IL-6, TGF-ß, RORγt, T-bet, Foxp-3 and GATA-3. RESULTS: The mRNA levels of TJ-associated genes were statistically significantly reduced soon after viral exposures and the mRNA levels of claudin-1, occludin and ZO-1 in viral positive tissues (from Group I) were lower than that in viral negative tissues (from Group II) after viral exposure. IL-17A mRNA levels were also decreased and positively correlated with the mRNA levels of the TJ-associated genes after viral exposure or infection, although the levels of IL-6, TGF-ß and RORγt mRNA showed no statistical difference. The levels of GATA-3 mRNA in tissues collected before viral exposure were statistically different between Group I and Group II animals. The balance between T-bet and GATA-3 mRNA levels in Group II was markedly altered and statistically significantly different from that in Group I. CONCLUSIONS: Acute SHIV, and by extension HIV infection could affect the expression of TJ-associated genes, probably through IL-17A and other immune alterations.

Mucosal addressin cell adhesion molecule-1 of rhesus macaques: molecular cloning, expression, and alteration after viral infection.

BACKGROUND: Mucosal addressin cell adhesion molecule-1 (MAdCAM-1), a member of the immunoglobulin superfamily, is essential for gut-specific homing of leukocytes; however, it has not been well characterized in rhesus macaques. AIMS: To obtain the complete nucleotide sequence of rhesus macaque MAdCAM-1 cDNA and determine its distribution in gut-associated lymphoid tissues (GALT) and its alteration in duodenal mucosa after simian/human immunodeficiency virus (SHIV) infection. METHODS: MAdCAM-1 cDNA was cloned from the colon mucosa of a rhesus macaque by 3'- and 5'-RACE. The distribution and abundance of MAdCAM-1 mRNA in the GALT were examined by nested and real-time RT-PCR. The alterations of MAdCAM-1 mRNA levels in SHIV-infected duodenal mucosa were determined by real-time RT-PCR. RESULTS: The nucleotide sequence of rhesus macaque MAdCAM-1 cDNA (1,503 bp nucleotides) including the 5'- and 3'-untranslated regions was obtained. The coding region (1,086 bp) showed 87.56% and the Ig-like domain 1, 2 and TM + cytoplasmic domains showed >93% nucleotide sequence identity to that of humans. Like humans, rhesus macaques lacked MAdCAM-1 IgA1-like domain, which could be a common feature for all primates appeared later during vertebrate evolution. Two species of MAdCAM-1 mRNA were detected and high-level transcripts were observed primarily in the GALT. The full-length MAdCAM-1 expressed in vitro could bind to human α4ß7. MAdCAM-1 mRNA levels were statistically significantly reduced in SHIV-infected duodenal mucosa. CONCLUSIONS: These data provided a basis for using rhesus macaques in pathological and therapeutic studies on leukocyte homing related diseases such as inflammatory bowel disease and HIV/AIDS.

Immunogenicity and safety of boosting with a recombinant two-component SARS-CoV-2 vaccine: two randomized, parallel-controlled, phase 2 studies.

BACKGROUND: Recombinant protein vaccines are vital for broad protection against SARS-CoV-2 variants. This study assessed ReCOV as a booster in two Phase 2 trials. RESEARCH DESIGN AND METHODS: Study-1 involved subjects were randomized (1:1:1) to receive 20 µg ReCOV, 40 µg ReCOV, or an inactivated vaccine (COVILO®) in the United Arab Emirates. Study-2 participating individuals were randomized (1:1:1) to receive 20 µg ReCOV (pilot batch, ReCOV HA), 20 µg ReCOV (commercial batch, ReCOV TC), or 30 µg BNT162b2 (COMIRNATY®) in the Philippines. The primary immunogenicity objectives was to compare the geometric mean titer (GMT) and seroconversion rate (SCR) of neutralizing antibodies induced by one ReCOV booster dose with those of inactivated vaccine and BNT162b2, respectively, at 14 days post-booster. RESULTS: Heterologous ReCOV booster doses were safe and induced comparable immune responses to inactivated vaccines and BNT162b2 against Omicron variants and the prototype. They showed significant advantages in cross-neutralization against multiple SARS-CoV-2 variants, surpassing inactivated vaccines and BNT162b2, with good immune persistence. CONCLUSIONS: Heterologous ReCOV boosting was safe and effective, showing promise in combating COVID-19. The study highlights ReCOV's potential for enhanced protection, supported by strong cross-neutralization and immune persistence. CLINICAL TRIAL REGISTRATION: Study-1, www.clinicaltrials.gov, identifier is NCT05323435; Study-2, www.clinicaltrials.gov, identifier is NCT05084989.

Investigation of the role of healthy dogs as potential carriers of rabies virus.

To investigate whether healthy animals are potential carriers of rabies virus in China, 153 domestic dogs were collected from a rabies enzootic area, Anlong county in Guizhou Province, and monitored for 6 months. Initially, findings of rabies virus antigen in the saliva of 15 dogs by an enzyme-linked immunosorbent assay (ELISA) test suggested they might be carriers. These 15 dogs were kept under observation for 6 months. None of the dogs showed any clinical signs of rabies during the observation period. Moreover, using the ELISA test alone, detection of rabies virus antigen in saliva of some animals was not consistent during the observation period. However, none of the saliva samples collected either at the time of acquisition or during the observation period was found to be positive for rabies virus RNA by reverse transcriptase-polymerase chain reaction (RT-PCR). Furthermore, neither viral antigen nor viral RNA was detected in the brain samples collected at the time of euthanasia. These results do not provide support for the contention that healthy dogs act as carriers in rabies. Caution is urged when preliminary and nondefinitive tests, such as ELISA, are used to infer clinical status related to rabies.

Molecular cloning and characterization of DNGR-1 in rhesus macaques.

DC, NK lectin group receptor-1 (DNGR-1), also known as C-type lectin domain family 9 member A (CLEC9A), is a promising target for immunological therapeutics and vaccination against tumors and viruses. However, little is known about its property in rhesus macaques. In this study, we cloned rhesus macaque DNGR-1 cDNA, and found that its coding region could encode a 241-amino acid polypeptide with 91.7% sequence identity and similar antigenicity to that of humans. Both free and cell surface rhesus macaque DNGR-1 expressed in vitro could bind to apoptotic/dead cells induced by serum deprivation or freeze-thaw, and to pyroptotic cells stimulated with PMA and LPS. We also demonstrated that rhesus macaque DNGR-1 mRNA was present in all the examined tissues, with the highest in lymph nodes, spleen, blood, and thymus. The expression of DNGR-1 that is highly similar to that of humans warranted the usefulness of rhesus macaques in testing human therapeutics and vaccines targeting DNGR-1, especially those for HIV/AIDS.

The levels of DNGR-1 and its ligand-bearing cells were altered after human and simian immunodeficiency virus infection.

Dendritic cell NK lectin Group Receptor-1 (DNGR-1), also known as C-type lectin domain family 9, member A (CLEC9A), is a member of C-type lectin receptor superfamily expressed primarily on dendritic cells (DC) that excel in cross-presentation of exogenous antigens. To find out whether and how it is affected in human immunodeficiency virus infections or acquired immunodeficiency syndromes (HIV/AIDS), DNGR-1 expression and DNGR-1-binding cells in simian/human immunodeficiency virus (SHIV) and simian immunodeficiency virus (SIV)-infected rhesus macaques and antiretroviral therapy (ART)-treated AIDS patients were examined by real-time RT-PCR, flow cytometry, and confocal microscopy. DNGR-1 expression was observed in both lymphoid and non-lymphoid tissues including gut-associated lymphoid tissues (GALT) of rhesus macaques. DNGR-1 mRNA levels were significantly reduced in the blood while significantly elevated in the GALT of SHIV/SIV-infected rhesus macaques. DNGR-1 transcription levels were also significantly reduced in the blood of ART-treated AIDS patients irrespective of viral status. White blood cells with exposed DNGR-1 ligands were significantly increased in ART-treated AIDS patients, while significantly decreased in SIV-infected rhesus macaques. These data indicate that DNGR-1 expression, and by extension, the function of cross-presentation of antigens associated with dead/damaged cells might be compromised in HIV/SIV infection, which might play a role in HIV/AIDS pathogenesis and should be taken into consideration in therapeutic AIDS vaccine development.

Expression of pIgR in the tracheal mucosa of SHIV/SIV-infected rhesus macaques.

Polymeric immunoglobulin receptors(pIgR) are key participants in the formation and secretion of secretory IgA(S-IgA), which is critical for the prevention of microbial infection and colonization in the respiratory system. Although increased respiratory colonization and infections are common in HIV/AIDS, little is known about the expression of pIgR in the airway mucosa of these patients. To address this, the expression levels of pIgR in the tracheal mucosa and lungs of SHIV/SIV-infected rhesus macaques were examined by real-time RTPCR and confocal microscopy. We found that the levels of both PIGR mRNA and pIgR immunoreactivity were lower in the tracheal mucosa of SHIV/SIV-infected rhesus macaques than that in non-infected rhesus macaques, and the difference in pIgR immunoreactivity was statistically significant. IL-17A, which enhances pIgR expression, was also changed in the same direction as that of pIgR. In contrast to changes in the tracheal mucosa, pIgR and IL-17A levels were higher in the lungs of infected rhesus macaques. These results indicated abnormal pIgR expression in SHIV/SIV, and by extension HIV infections, which might partially result from IL-17A alterations and might contribute to the increased microbial colonization and infection related to pulmonary complications in HIV/AIDS.

[Cloning and expression of the nucleoprotein gene of Puumala-like virus].

OBJECTIVE: In order to detect Hokkaido virus (HOKV), a recombinant baculovirus containing the nucleoprotein (NP) gene of HOKV was constructed, and then the NP was expressed in insect cell. METHODS: The NP gene was cloned into plasmid PCR 2.1TA vector and then was ligated into baculovirus donor plasmid pFastBac after cutting by the restriction enzyme Kpn I and Not I. pFastBac 1 was subsequently transferred into the One Short TOP10 competent cells and then into DH1OBac E. coli competent cells, which contained the baculovirus shuttle vector (Bacmid) and the helper plasmid to generate a recombinant bacmid. RESULTS: The NP gene was successfully expressed in Sf9 insect cell. The expressed recombinant nucleoprotein had been identified in the Sf9 insect cell by indirect immunofluorescence assay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. The results showed that the recombinant nucleoprotein appeared a molecular weight of 50 x 10(3) Mr, and could reacted with anti-recombinant Puumala virus (PUUV) nucleocapsid monoclonal antibodies and polyclonal antibodies against hantavirus. CONCLUSION: Our results indicated that the recombinant nucleoprotein was successfully expressed and having the immunogenicity and reactivity of natural nucleoprotein of HOKV.