Current design of Zika virus (ZIKV) vaccine mainly considered envelope (E) as the major target antigen. Non-structural protein NS1 was seldom considered. Herein, we generated three adenovirus-vectored vaccines carrying E (Ad2-E), or premembrane/membrane (prM/M) with E (Ad2-prME), or NS1 in addition to prM/M with E (Ad2-prME-NS1). Ad2-prME induced higher neutralizing antibody response to ZIKV than Ad2-E, suggesting prM/M is important for the folding of immunogenic E. Most intriguingly, Ad2-prME-NS1 elicited the best viral inhibition when the immune sera were added to ZIKV-infected cells. In ZIKV-challenged neonatal mice born to maternally immunized dams, Ad2-prME-NS1 conferred the best protection in preventing weight loss, neurological disorders, and viral replication. Ad2-prME also conferred significant protection but was less effective than Ad2-prME-NS1, whereas Ad2-E only alleviated neurological symptoms but did not inhibit viral replication. Our study suggested that NS1 should be considered in the design of ZIKV vaccine in addition to prM/M and E.

Human adenovirus type 55 (HAdV55) represents an emerging respiratory pathogen and causes severe pneumonia with high fatality in humans. The cellular receptors, which are essential for understanding the infection and pathogenesis of HAdV55, remain unclear. Here, we found that HAdV55 binding and infection were sharply reduced by disrupting the interaction of viral fiber protein with human desmoglein-2 (hDSG2) but only slightly reduced by disrupting the interaction of viral fiber protein with human CD46 (hCD46). Loss-of-function studies using soluble receptors, blocking antibodies, RNA interference, and gene knockout demonstrated that hDSG2 predominantly mediated HAdV55 infection. Non-permissive rodent cells became susceptible to HAdV55 infection when hDSG2 or hCD46 was expressed, but hDSG2 mediated more efficient HAd55 infection than hCD46. We generated two transgenic mouse lines that constitutively express either hDSG2 or hCD46. Although non-transgenic mice were resistant to HAdV55 infection, infection of HAd55 was significantly increased in hDSG2+/+ mice but was much less increased in hCD46+/+ mice. Our findings demonstrate that both hDSG2 and hCD46 are able to mediate HAdV55 infection but hDSG2 plays the major roles. The hDSG2 transgenic mouse can be used as a rodent model for evaluation of HAdV55 vaccine and therapeutics. IMPORTANCE Human adenovirus type 55 (HAdV55) has recently emerged as a highly virulent respiratory pathogen and has been linked to severe and even fatal pneumonia in immuno-competent adults. However, the cellular receptors mediating the entry of HAdV55 into host cells remain unclear, which hinders the establishment of HAdV55-infected animal models and the development of antiviral approaches. In this study, we demonstrated that human desmoglein-2 (hDSG2) plays the major roles during HAdV55 infection. Human CD46 (hCD46) could also mediate the infection of HAdV55, but the efficiency was much lower than hDSG2. We generated two transgenic mouse lines that express either hDSG2 or hCD46, both of which enabled HAd55 infection in otherwise non-transgenic mice. hDSG2 transgenic mice enabled more efficient HAdV55 infection than hCD46 transgenic mice. Our study adds to our understanding of HAdV55 infection and provides an animal model for evaluating HAdV55 vaccines and therapeutics.

Zika virus (ZIKV) infection during pregnancy causes congenital defects such as fetal microcephaly. Monoclonal antibodies (MAbs) against the nonstructural protein 1 (NS1) have the potential to suppress ZIKV pathogenicity without enhancement of disease, but the pathways through which they confer protection remain obscure. Here, we report two types of NS1-targeted human MAbs that inhibit ZIKV infection through distinct mechanisms. MAbs 3G2 and 4B8 show a better efficacy than MAb 4F10 in suppressing ZIKV infection in C57BL/6 neonatal mice. Unlike MAb 4F10 that mainly triggers antibody-dependent cell-mediated cytotoxicity (ADCC), MAbs 3G2 and 4B8 not only trigger ADCC but inhibit ZIKV infection without Fcγ receptor-bearing effector cells, possibly at postentry stages. Destroying the Fc-mediated effector function of MAbs 3G2 and 4B8 reduces but does not abolish their protective effects, whereas destroying the effector function of MAb 4F10 eliminates the protective effects, suggesting that MAbs 3G2 and 4B8 engage both Fcγ receptor-dependent and -independent pathways. Further analysis reveals that MAbs 3G2 and 4B8 target the N-terminal region of NS1 protein, whereas MAb 4F10 targets the C-terminal region, implying that the protective efficacy of an NS1-targeted MAb may be associated with its epitope recognition. Our results illustrate that NS1-targeted MAbs have multifaceted protective effects and provide insights for the development of NS1-based vaccines and therapeutics. IMPORTANCE Zika virus (ZIKV) is a mosquito-borne flavivirus that has been linked to congenital microcephaly during recent epidemics. No licensed antiviral drug or vaccine is available. Monoclonal antibodies (MAbs) against the nonstructural protein 1 (NS1) inhibit ZIKV pathogenicity but do not enhance the disease as envelope protein-targeted MAbs do. However, the protection mechanisms are not fully understood. Here, we show that in the presence or absence of Fcγ receptor-bearing effector cells, NS1-targeted human MAbs 3G2 and 4B8 inhibit ZIKV infection. Compared to MAb 4F10 that has no inhibitory effects without effector cells, 3G2 and 4B8 confer better protection in ZIKV-infected neonatal mice. Destroying the Fc-mediated effector function reduces but does not abolish the protection of 3G2 and 4B8, suggesting that they engage both Fcγ receptor-dependent and -independent pathways. The protective efficacy of NS1-targeted MAbs may be associated with their epitope recognition. Our findings will help to develop NS1-based vaccines and therapeutics.

SummaryThe genes encoding the b-and b8-subunits of RNA polymerase (rpoB and rpoC respectively) are fused as one continuous open reading frame in Helicobacter pylori and in other members of this genus, but are separate in other bacterial taxonomic groups, including the closely related genus Campylobacter. To test whether this b±b8 tethering is essential, we used polymerase chain reaction-based cloning to separate the rpoB and rpoC moieties of the H. pylori rpoB ±rpoC fusion gene with a non-polar chloramphenicol resistance cassette containing a new translational start, and introduced this construct into H. pylori by electrotransformation. H. pylori containing these separated rpoB and rpoC genes in place of the native fusion gene produced non-tethered b and b8 RNAP subunits, grew well in culture and colonized and proliferated well in conventional C57BL/6 mice. Thus, the extraordinary b±b8 tethering is not essential for H. pylori viability and gastric colonization.

Human adenoviruses type 4 (HAdV4) and 7 (HAdV7) are two major respiratory pathogens and sporadically cause outbreaks of acute respiratory diseases. The neutralizing antibody (nAb) response to these two adenoviruses in civilian populations, which is important for dissecting previous circulations and predicting potential outbreaks, remains largely unknown. In this study, we generated replication-competent HAdV4 and HAdV7 reporter viruses expressing secreted-alkaline-phosphatase (SEAP), and established neutralization assays to investigate the seroprevalence of pre-existing nAb in healthy volunteers from Hunan Province, southern China. The seropositivity rates are 58.4 and 63.8% for anti-HAdV4 nAb and anti-HAdV7 nAb, respectively. High nAb titers (> 1000) were frequently detected in HAdV4-seropositive individuals, whereas most HAdV7-seropositive volunteers had moderate nAb titers (201–1000). The seropositivity rates of anti-HAdV4 nAb and anti-HAdV7 nAb increase with age, with individuals younger than 20 exhibiting the lowest seropositivity rates. Both seropositivity rates and nAb titers are comparable between different sex groups. Notably, HAdV4-seropositive individuals tend to be HAdV7-seropositive and vice versa. Because HAdV4 antisera showed no neutralizing activity to HAdV7 whereas HAdV7 antisera cannot neutralize HAdV4, a subgroup of individuals might be susceptible to infection by HAdV4 and HAdV7 and thus generate nAb to both of them. These results revealed the continuous circulation of HAdV4 and HAdV7 and the lack of protective immunity in more than 35% of people, which emphasized the surveillance of these two HAdVs and the development of prophylactic vaccines.