Intradermal but not intramuscular modified vaccinia Ankara immunizations protect against intravaginal tier2 simian-human immunodeficiency virus challenges in female macaques.

Route of immunization can markedly influence the quality of immune response. Here, we show that intradermal (ID) but not intramuscular (IM) modified vaccinia Ankara (MVA) vaccinations provide protection from acquisition of intravaginal tier2 simian-human immunodeficiency virus (SHIV) challenges in female macaques. Both routes of vaccination induce comparable levels of serum IgG with neutralizing and non-neutralizing activities. The protection in MVA-ID group correlates positively with serum neutralizing and antibody-dependent phagocytic activities, and envelope-specific vaginal IgA; while the limited protection in MVA-IM group correlates only with serum neutralizing activity. MVA-ID immunizations induce greater germinal center Tfh and B cell responses, reduced the ratio of Th1 to Tfh cells in blood and showed lower activation of intermediate monocytes and inflammasome compared to MVA-IM immunizations. This lower innate activation correlates negatively with induction of Tfh responses. These data demonstrate that the MVA-ID vaccinations protect against intravaginal SHIV challenges by modulating the innate and T helper responses.

A microbiota and dietary metabolite integrates DNA repair and cell death to regulate embryo viability and aneuploidy during aging.

During aging, environmental stressors and mutations along with reduced DNA repair cause germ cell aneuploidy and genome instability, which limits fertility and embryo development. Benevolent commensal microbiota and dietary plants secrete indoles, which improve healthspan and reproductive success, suggesting regulation of germ cell quality. We show that indoles prevent aneuploidy and promote DNA repair and embryo viability, which depends on age and genotoxic stress levels and affects embryo quality across generations. In young animals or with low doses of radiation, indoles promote DNA repair and embryo viability; however, in older animals or with high doses of radiation, indoles promote death of the embryo. These studies reveal a previously unknown quality control mechanism by which indole integrates DNA repair and cell death responses to preclude germ cell aneuploidy and ensure transgenerational genome integrity. Such regulation affects healthy aging, reproductive senescence, cancer, and the evolution of genetic diversity in invertebrates and vertebrates.

V2 hotspot optimized MVA vaccine expressing stabilized HIV-1 Clade C envelope Gp140 delays acquisition of heterologous Clade C Tier 2 challenges in Mamu-A*01 negative Rhesus Macaques.

Stabilized HIV envelope (Env) trimeric protein immunogens have been shown to induce strong autologous neutralizing antibody response. However, there is limited data on the immunogenicity and efficacy of stabilized Env expressed by a viral vector-based immunogen. Here, we compared the immunogenicity and efficacy of two modified vaccinia Ankara (MVA) vaccines based on variable loop 2 hotspot (V2 HS) optimized C.1086 envelope (Env) sequences, one expressing the membrane anchored gp150 (MVA-150) and the other expressing soluble uncleaved pre-fusion optimized (UFO) gp140 trimer (MVA-UFO) in a DNA prime/MVA boost approach against heterologous tier 2 SHIV1157ipd3N4 intrarectal challenges in rhesus macaques (RMs). Both MVA vaccines also expressed SIVmac239 Gag and form virus-like particles. The DNA vaccine expressed SIVmac239 Gag, C.1086 gp160 Env and rhesus CD40L as a built-in adjuvant. Additionally, all immunizations were administered intradermally (ID) to reduce induction of vaccine-specific IFNγ+ CD4 T cell responses. Our results showed that both MVA-150 and MVA-UFO vaccines induce comparable Env specific IgG responses in serum and rectal secretions. The vaccine-induced serum antibody showed ADCC and ADCVI activities against the challenge virus. Comparison with a previous study that used similar immunogens via intramuscular route (IM) showed that ID immunizations induced markedly lower SHIV specific CD4 and CD8 T cell responses compared to IM immunizations. Following challenge, MVA-UFO vaccinated animals showed a significant delay in acquisition of SHIV1157ipd3N4 infection but only in Mamu-A*01 negative macaques with an estimated vaccine efficacy of 64% per exposure. The MVA-150 group also showed a trend (p=0.1) for delay in acquisition of SHIV infection with an estimated vaccine efficacy of 57%. The vaccine-induced IFNγ secreting CD8 T cell responses showed a direct association and CD4 T cells showed an inverse association with delay in acquisition of SHIV infection. These results demonstrated that both MVA-150 and MVA-UFO immunogens induce comparable humoral and cellular immunity and the latter provides marginally better protection against heterologous tier 2 SHIV infection. They also demonstrate that DNA/MVA vaccinations delivered by ID route induce better antibody and lower CD4 and CD8 T cell responses compared to IM.

A clade C HIV-1 vaccine protects against heterologous SHIV infection by modulating IgG glycosylation and T helper response in macaques.

The rising global HIV-1 burden urgently requires vaccines capable of providing heterologous protection. Here, we developed a clade C HIV-1 vaccine consisting of priming with modified vaccinia Ankara (MVA) and boosting with cyclically permuted trimeric gp120 (CycP-gp120) protein, delivered either orally using a needle-free injector or through parenteral injection. We tested protective efficacy of the vaccine against intrarectal challenges with a pathogenic heterologous clade C SHIV infection in rhesus macaques. Both routes of vaccination induced a strong envelope-specific IgG in serum and rectal secretions directed against V1V2 scaffolds from a global panel of viruses with polyfunctional activities. Envelope-specific IgG showed lower fucosylation compared with total IgG at baseline, and most of the vaccine-induced proliferating blood CD4+ T cells did not express CCR5 and α4ß7, markers associated with HIV target cells. After SHIV challenge, both routes of vaccination conferred significant and equivalent protection, with 40% of animals remaining uninfected at the end of six weekly repeated challenges with an estimated efficacy of 68% per exposure. Induction of envelope-specific IgG correlated positively with G1FB glycosylation, and G2S2F glycosylation correlated negatively with protection. Vaccine-induced TNF-α+ IFN-γ+ CD8+ T cells and TNF-α+ CD4+ T cells expressing low levels of CCR5 in the rectum at prechallenge were associated with decreased risk of SHIV acquisition. These results demonstrate that the clade C MVA/CycP-gp120 vaccine provides heterologous protection against a tier2 SHIV rectal challenge by inducing a polyfunctional antibody response with distinct Fc glycosylation profile, as well as cytotoxic CD8 T cell response and CCR5-negative T helper response in the rectum.

Structure-guided changes at the V2 apex of HIV-1 clade C trimer enhance elicitation of autologous neutralizing and broad V1V2-scaffold antibodies.

HIV-1 clade C envelope immunogens that elicit both neutralizing and non-neutralizing V1V2-scaffold-specific antibodies (protective correlates from RV144 human trial) are urgently needed due to the prevalence of this clade in the most impacted regions worldwide. To achieve this, we introduce structure-guided changes followed by consensus-C-sequence-guided optimizations at the V2 region to generate UFO-v2-RQH173 trimer. This improves the abundance of well-formed trimers. Following the immunization of rabbits, the wild-type protein fails to elicit any autologous neutralizing antibodies, but UFO-v2-RQH173 elicits both autologous neutralizing and broad V1V2-scaffold antibodies. The variant with a 173Y modification in the V2 region, most prevalent among HIV-1 sequences, shows decreased ability in displaying a native-like V1V2 epitope with time in vitro and elicited antibodies with lower neutralizing and higher V1V2-scaffold activities. Our results identify a stabilized clade C trimer capable of eliciting improved neutralizing and V1V2-scaffold antibodies and reveal the importance of the V2 region in tuning this.

SARS-CoV-2 RBD trimer protein adjuvanted with Alum-3M-052 protects from SARS-CoV-2 infection and immune pathology in the lung.

There is a great need for the development of vaccines that induce potent and long-lasting protective immunity against SARS-CoV-2. Multimeric display of the antigen combined with potent adjuvant can enhance the potency and longevity of the antibody response. The receptor binding domain (RBD) of the spike protein is a primary target of neutralizing antibodies. Here, we developed a trimeric form of the RBD and show that it induces a potent neutralizing antibody response against live virus with diverse effector functions and provides protection against SARS-CoV-2 challenge in mice and rhesus macaques. The trimeric form induces higher neutralizing antibody titer compared to monomer with as low as 1µg antigen dose. In mice, adjuvanting the protein with a TLR7/8 agonist formulation alum-3M-052 induces 100-fold higher neutralizing antibody titer and superior protection from infection compared to alum. SARS-CoV-2 infection causes significant loss of innate cells and pathology in the lung, and vaccination protects from changes in innate cells and lung pathology. These results demonstrate RBD trimer protein as a suitable candidate for vaccine against SARS-CoV-2.

A modified vaccinia Ankara vector-based vaccine protects macaques from SARS-CoV-2 infection, immune pathology, and dysfunction in the lungs.

A combination of vaccination approaches will likely be necessary to fully control the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Here, we show that modified vaccinia Ankara (MVA) vectors expressing membrane-anchored pre-fusion stabilized spike (MVA/S) but not secreted S1 induced strong neutralizing antibody responses against SARS-CoV-2 in mice. In macaques, the MVA/S vaccination induced strong neutralizing antibodies and CD8+ T cell responses, and conferred protection from SARS-CoV-2 infection and virus replication in the lungs as early as day 2 following intranasal and intratracheal challenge. Single-cell RNA sequencing analysis of lung cells on day 4 after infection revealed that MVA/S vaccination also protected macaques from infection-induced inflammation and B cell abnormalities and lowered induction of interferon-stimulated genes. These results demonstrate that MVA/S vaccination induces neutralizing antibodies and CD8+ T cells in the blood and lungs and is a potential vaccine candidate for SARS-CoV-2.

Protein model discrimination attempts using mutational sensitivity, predicted secondary structure, and model quality information.

Structure prediction methods often generate a large number of models for a target sequence. Even if the correct fold for the target sequence is sampled in this dataset, it is difficult to distinguish it from other decoy structures. An attempt to solve this problem using experimental mutational sensitivity data for the CcdB protein was described previously by exploiting the correlation of residue depth with mutational sensitivity (r ~ 0.6). We now show that such a correlation extends to four other proteins with localized active sites, and for which saturation mutagenesis datasets exist. We also examine whether incorporation of predicted secondary structure information and the DOPE model quality assessment score, in addition to mutational sensitivity, improves the accuracy of model discrimination using a decoy dataset of 163 targets from CASP. Although most CASP models would have been subjected to model quality assessment prior to submission, we find that the DOPE score makes a substantial contribution to the observed improvement. We therefore also applied the approach to CcdB and four other proteins for which reliable experimental mutational data exist and observe that inclusion of experimental mutational data results in a small qualitative improvement in model discrimination relative to that seen with just the DOPE score. This is largely because of our limited ability to quantitatively predict effects of point mutations on in vivo protein activity. Further improvements in the methodology are required to facilitate improved utilization of single mutant data.

Indoles from commensal bacteria extend healthspan.

Multiple studies have identified conserved genetic pathways and small molecules associated with extension of lifespan in diverse organisms. However, extending lifespan does not result in concomitant extension in healthspan, defined as the proportion of time that an animal remains healthy and free of age-related infirmities. Rather, mutations that extend lifespan often reduce healthspan and increase frailty. The question arises as to whether factors or mechanisms exist that uncouple these processes and extend healthspan and reduce frailty independent of lifespan. We show that indoles from commensal microbiota extend healthspan of diverse organisms, including Caenorhabditis elegans, Drosophila melanogaster, and mice, but have a negligible effect on maximal lifespan. Effects of indoles on healthspan in worms and flies depend upon the aryl hydrocarbon receptor (AHR), a conserved detector of xenobiotic small molecules. In C. elegans, indole induces a gene expression profile in aged animals reminiscent of that seen in the young, but which is distinct from that associated with normal aging. Moreover, in older animals, indole induces genes associated with oogenesis and, accordingly, extends fecundity and reproductive span. Together, these data suggest that small molecules related to indole and derived from commensal microbiota act in diverse phyla via conserved molecular pathways to promote healthy aging. These data raise the possibility of developing therapeutics based on microbiota-derived indole or its derivatives to extend healthspan and reduce frailty in humans.

Residue proximity information and protein model discrimination using saturation-suppressor mutagenesis.

Identification of residue-residue contacts from primary sequence can be used to guide protein structure prediction. Using Escherichia coli CcdB as the test case, we describe an experimental method termed saturation-suppressor mutagenesis to acquire residue contact information. In this methodology, for each of five inactive CcdB mutants, exhaustive screens for suppressors were performed. Proximal suppressors were accurately discriminated from distal suppressors based on their phenotypes when present as single mutants. Experimentally identified putative proximal pairs formed spatial constraints to recover >98% of native-like models of CcdB from a decoy dataset. Suppressor methodology was also applied to the integral membrane protein, diacylglycerol kinase A where the structures determined by X-ray crystallography and NMR were significantly different. Suppressor as well as sequence co-variation data clearly point to the X-ray structure being the functional one adopted in vivo. The methodology is applicable to any macromolecular system for which a convenient phenotypic assay exists.

Protein model discrimination using mutational sensitivity derived from deep sequencing.

A major bottleneck in protein structure prediction is the selection of correct models from a pool of decoys. Relative activities of ∼1,200 individual single-site mutants in a saturation library of the bacterial toxin CcdB were estimated by determining their relative populations using deep sequencing. This phenotypic information was used to define an empirical score for each residue (RankScore), which correlated with the residue depth, and identify active-site residues. Using these correlations, ∼98% of correct models of CcdB (RMSD ≤ 4Å) were identified from a large set of decoys. The model-discrimination methodology was further validated on eleven different monomeric proteins using simulated RankScore values. The methodology is also a rapid, accurate way to obtain relative activities of each mutant in a large pool and derive sequence-structure-function relationships without protein isolation or characterization. It can be applied to any system in which mutational effects can be monitored by a phenotypic readout.