Recognition of a highly conserved glycoprotein B epitope by a bivalent antibody neutralizing HCMV at a post-attachment step.

Human cytomegalovirus (HCMV) is one of the main causative agents of congenital viral infection in neonates. HCMV infection also causes serious morbidity and mortality among organ transplant patients. Glycoprotein B (gB) is a major target for HCMV neutralizing antibodies, yet the underlying neutralization mechanisms remain largely unknown. Here we report that 3-25, a gB-specific monoclonal antibody previously isolated from a healthy HCMV-positive donor, efficiently neutralized 14 HCMV strains in both ARPE-19 cells and MRC-5 cells. The core epitope of 3-25 was mapped to a highly conserved linear epitope on antigenic domain 2 (AD-2) of gB. A 1.8 Å crystal structure of 3-25 Fab in complex with the peptide epitope revealed the molecular determinants of 3-25 binding to gB at atomic resolution. Negative-staining electron microscopy (EM) 3D reconstruction of 3-25 Fab in complex with de-glycosylated postfusion gB showed that 3-25 Fab fully occupied the gB trimer at the N-terminus with flexible binding angles. Functionally, 3-25 efficiently inhibited HCMV infection at a post-attachment step by interfering with viral membrane fusion, and restricted post-infection viral spreading in ARPE-19 cells. Interestingly, bivalency was required for HCMV neutralization by AD-2 specific antibody 3-25 but not the AD-4 specific antibody LJP538. In contrast, bivalency was not required for HCMV binding by both antibodies. Taken together, our results reveal the structural basis of gB recognition by 3-25 and demonstrate that inhibition of viral membrane fusion and a requirement of bivalency may be common for gB AD-2 specific neutralizing antibody.

Functional Evaluation and Genetic Evolution of Human T-Cell Responses After Vaccination With a Conditionally Replication-Defective Cytomegalovirus Vaccine.

BACKGROUND: Cytomegalovirus (CMV) can cause congenital infection and is the leading cause of nongenetic newborn disabilities. V160, a conditionally replication-defective virus, is an investigational vaccine under evaluation for prevention of congenital CMV. The vaccine was well tolerated and induced both humoral and cellular immunity in CMV-seronegative trial participants. T-cell-mediated immunity is important for immune control of CMV. Here we describe efforts to understand the quality attributes of the T-cell responses induced by vaccination. METHODS: Using multicolor flow cytometry, we analyzed vaccine-induced T cells for memory phenotype, antigen specificity, cytokine profiles, and cytolytic potential. Moreover, antigen-specific T cells were sorted from 4 participants, and next-generation sequencing was used to trace clonal lineage development during the course of vaccination using T-cell receptor ß-chain sequences as identifiers. RESULTS: The results demonstrated that vaccination elicited polyfunctional CD4 and CD8 T cells to 2 dominant antigens, pp65 and IE1, with a predominantly effector phenotype. Analysis of T-cell receptor repertoires showed polyclonal expansion of pp65- and IE1-specific T cells after vaccination. CONCLUSION: V160 induced a genetically diverse and polyfunctional T-cell response and the data support further clinical development of V160 for prevention of CMV infection and congenital transmission. CLINICAL TRIALS REGISTRATION: NCT01986010.

Potent Bispecific Neutralizing Antibody Targeting Glycoprotein B and the gH/gL/pUL128/130/131 Complex of Human Cytomegalovirus.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that can cause developmental disorders following congenital infection and life-threatening complications among transplant patients. Potent neutralizing monoclonal antibodies (MAbs) are promising drug candidates against HCMV infection. HCMV can infect a broad range of cell types. Therefore, single neutralizing antibodies targeting one HCMV glycoprotein often lack either potency or broad cell-type coverage. We previously characterized two human-derived HCMV neutralizing MAbs. One was the broadly neutralizing MAb 3-25, which targets the antigenic domain 2 of glycoprotein B (gB). The other was the highly potent MAb 2-18, which specifically recognizes the gH/gL/pUL128/130/131 complex (pentamer). To combine the strengths of gB- and pentamer-targeting MAbs, we developed an IgG-single-chain variable fragment (scFv) bispecific antibody by fusing the 2-18 scFv to the heavy-chain C terminus of MAb 3-25. The resulting bispecific antibody showed high-affinity binding to both gB and pentamer. Functionally, the bispecific antibody demonstrated a combined neutralization breadth and potency of the parental MAbs in multiple cell lines and inhibited postinfection viral spreading. Furthermore, the bispecific antibody was easily produced in CHO cells at a yield above 1 g/liter and showed a single-dose pharmacokinetic profile comparable to that of parental MAb 3-25 in rhesus macaques. Importantly, the bispecific antibody retained broadly and potent neutralizing activity after 21 days in circulation. Taken together, our research provides a proof-of-concept study for developing bispecific neutralizing antibody therapies against HCMV infection.

Identification of adipocyte plasma membrane-associated protein as a novel modulator of human cytomegalovirus infection.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that can cause disability in newborns and serious clinical diseases in immunocompromised patients. HCMV has a large genome with enormous coding potential; its viral particles are equipped with complicated glycoprotein complexes and can infect a wide range of human cells. Although multiple host cellular receptors interacting with viral glycoproteins have been reported, the mechanism of HCMV infection remains a mystery. Here we report identification of adipocyte plasma membrane-associated protein (APMAP) as a novel modulator active in the early stage of HCMV infection. APMAP is necessary for HCMV infection in both epithelial cells and fibroblasts; knockdown of APMAP expression significantly reduced HCMV infection of these cells. Interestingly, ectopic expression of human APMAP in cells refractory to HCMV infection, such as canine MDCK and murine NIH/3T3 cells, promoted HCMV infection. Furthermore, reduction in viral immediate early (IE) gene transcription at 6 h post infection and delayed nucleus translocation of tegument delivered pp65 at 4 h post infection were detected in APMAP-deficient cells but not in the wildtype cells. These results suggest that APMAP plays a role in the early stage of HCMV infection. Results from biochemical studies of APMAP and HCMV proteins suggest that APMAP could participate in HCMV infection through interaction with gH/gL containing glycoprotein complexes at low pH and mediate nucleus translocation of tegument pp65. Taken together, our results suggest that APMAP functions as a modulator promoting HCMV infection in multiple cell types and is an important player in the complex HCMV infection mechanism.

Identification of Variable and Joining Germline Genes and Alleles for Rhesus Macaque from B Cell Receptor Repertoires.

The rhesus macaque is a valuable preclinical animal model to estimate vaccine effectiveness and is also important for understanding Ab maturation and B cell repertoire evolution responding to vaccination. However, incomplete mapping of rhesus Ig germline genes hinders the research efforts. To address this deficiency, we sequenced the BCR repertoires of 75 Indian rhesus macaques. Using a bioinformatic method that has been validated with BCR repertoire analysis of three human donors, we were able to infer rhesus variable (V) and joint (J) germline alleles. We identified a total of 122 V and 20 J germline alleles, of which 91 V and 13 J alleles were novel, with 40 V novel genes, of which 8 were located at a novel genomic region not, to our knowledge, previously recorded. The novelty of these newly identified alleles was supported by two observations. First, the 50 V and 5 J novel alleles were observed in the whole genome sequencing data of 10 rhesus macaques. Second, using alignment reference including the novel alleles, the mutation rate of the rearranged repertoires significantly declined in nine other irrelevant samples, and all our identified novel V and J alleles were 100%-identity mapped by rearranged repertoire data. These identified novel alleles, along with the previously reported alleles, provide an important reference for future investigations of rhesus immune repertoire evolution in response to vaccination or infection. In addition, the method outlined in our study offers a powerful foundation for the identification of novel Ig alleles in the future.

A Replication-Defective Human Cytomegalovirus Vaccine Elicits Humoral Immune Responses Analogous to Those with Natural Infection.

Human cytomegalovirus (HCMV) can cause congenital infections, which are a leading cause of childhood disabilities. Since the rate of maternal-fetal transmission is much lower in naturally infected (HCMV-seropositive) women, we hypothesize that a vaccine candidate capable of eliciting immune responses analogous to those of HCMV-seropositive subjects may confer protection against congenital HCMV. We have previously described a replication-defective virus vaccine based on strain AD169 (D. Wang, D. C. Freed, X. He, F. Li, et al., Sci Transl Med 8:362ra145, 2016, https://doi.org/10.1126/scitranslmed.aaf9387). The vaccine, named V160, has been shown to be safe and immunogenic in HCMV-seronegative human subjects, eliciting both humoral and cellular immune responses (S. P. Adler, S. E. Starr, S. A. Plotkin, S. H. Hempfling, et al., J Infect Dis 220:411-419, 2019, https://doi.org/10.1093/infdis/171.1.26). Here, we further showed that sera from V160-immunized HCMV-seronegative subjects have attributes similar in quality to those from seropositive subjects, including high-avidity antibodies to viral antigens, coverage against a panel of genetically distinct clinical isolates, and protection against viral infection in diverse types of human cells in culture. More importantly, vaccination appeared efficient in priming the human immune system, inducing memory B cells in six V160 recipients at frequencies comparable to those of three HCMV-seropositive subjects. Our results demonstrate the ability of V160 to induce robust and durable humoral memory responses to HCMV, justifying further clinical evaluation of the vaccine against congenital HCMV.IMPORTANCEIn utero HCMV infection can lead to miscarriage or childhood disabilities, and an effective vaccine is urgently needed. Since children born to women who are seropositive prior to pregnancy are less likely to be affected by congenital HCMV infection, it has been hypothesized that a vaccine capable of inducing an immune response resembling the responses in HCMV-seropositive women may be effective. We previously described a replication-defective virus vaccine that has been demonstrated safe and immunogenic in HCMV-seronegative subjects. Here, we conducted additional analyses to show that the vaccine can induce antibodies with functional attributes similar to those from HCMV-seropositive subjects. Importantly, vaccination can induce long-lived memory B cells at frequencies comparable to those seen in HCMV-seropositive subjects. We conclude that this vaccine is a promising candidate that warrants further clinical evaluation for prevention of congenital HCMV.

Comparative neutralizing potencies of antibodies suggest conservation as well as mechanistic differences in human cytomegalovirus entry into epithelial and endothelial cells.

Antibody neutralization of cytomegalovirus (CMV) entry into diverse cell types is a key consideration for development of vaccines and immunotherapeutics. CMV entry into fibroblasts differs significantly from entry into epithelial or endothelial cells: fibroblast entry is mediated by gB and gH/gL/gO, whereas both epithelial and endothelial cell entry require an additional pentameric complex (PC) comprised of gH/gL/UL128/UL130/UL131A. Because PC-specific antibodies in CMV-seropositive human sera do not affect fibroblast entry but potently block entry into epithelial or endothelial cells, substantially higher neutralizing potencies for CMV-positive sera are observed when assayed using epithelial cells as targets than when using fibroblasts. That certain sera exhibit similar discordances between neutralizing potencies measured using epithelial vs. endothelial cells (Gerna G. et al.J Gen Virol, 89:853-865, 2008) suggested that additional mechanistic differences may also exist between epithelial and endothelial cell entry. To further explore this issue, neutralizing potencies using epithelial and endothelial cells were simultaneously determined for eight CMV-positive human sera, CMV-hyperimmune globulin, and a panel of monoclonal or anti-peptide antibodies targeting specific epitopes in gB, gH, gH/gL, or the PC. No significant differences were observed between epithelial and endothelial neutralizing potencies of epitope-specific antibodies, CMV-hyperimmune globulin, or seven of the eight human sera. However, one human serum exhibited a six-fold higher potency for neutralizing entry into epithelial cells vs. endothelial cells. These results suggest that epitopes exist that are important for epithelial entry but are less critical, or perhaps dispensable, for endothelial cell entry. Their existence should be considered when developing monoclonal antibody therapies or subunit vaccines representing limited epitopes.

Phase 1 Clinical Trial of a Conditionally Replication-Defective Human Cytomegalovirus (CMV) Vaccine in CMV-Seronegative Subjects.

BACKGROUND: A conditionally replication-defective human cytomegalovirus (CMV) vaccine (V160) derived from AD169 and genetically engineered to express CMV pentameric complex (gH/gL/pUL128/pUL130/pUL131) was developed and evaluated for phase 1 vaccine safety and immunogenicity in CMV-seronegative and CMV-seropositive adults. METHODS: Subjects received 3 doses of V160 or placebo on day 1, month 1, and month 6. Four vaccine dose levels, formulated with or without aluminum phosphate adjuvant, were evaluated. Injection-site and systemic adverse events (AEs) and vaccine viral shedding were monitored. CMV-specific cellular and humoral responses were measured by interferon-gamma ELISPOT and virus neutralization assay up to 12 months after last dose. RESULTS: V160 was generally well-tolerated, with no serious AEs observed. Transient, mild-to-moderate injection-site and systemic AEs were reported more frequently in vaccinated subjects than placebo. Vaccine viral shedding was not detected in any subject, confirming the nonreplicating feature of V160. Robust neutralizing antibody titers were elicited and maintained through 12 months postvaccination. Cellular responses to structural and nonstructural viral proteins were observed, indicating de novo expression of viral genes postvaccination. CONCLUSIONS: V160 displayed an acceptable safety profile. Levels of neutralizing antibodies and T-cell responses in CMV-seronegative subjects were within ranges observed following natural CMV infection. CLINICAL TRIAL REGISTRATION: . NCT01986010.

Targeting Human-Cytomegalovirus-Infected Cells by Redirecting T Cells Using an Anti-CD3/Anti-Glycoprotein B Bispecific Antibody.

The host immune response to human cytomegalovirus (HCMV) is effective against HCMV reactivation from latency, though not sufficient to clear the virus. T cells are primarily responsible for the control of viral reactivation. When the host immune system is compromised, as in transplant recipients with immunosuppression, HCMV reactivation and progressive infection can cause serious morbidity and mortality. Adoptive T cell therapy is effective for the control of HCMV infection in transplant recipients. However, it is a highly personalized therapeutic regimen and is difficult to implement in routine clinical practice. In this study, we explored a bispecific-antibody strategy to direct non-HCMV-specific T cells to recognize and exert effector functions against HCMV-infected cells. Using a knobs-into-holes strategy, we constructed a bispecific antibody in which one arm is specific for CD3 and can trigger T cell activation, while the other arm, specific for HCMV glycoprotein B (gB), recognizes and marks HCMV-infected cells based on the expression of viral gB on their surfaces. We showed that this bispecific antibody was able to redirect T cells with specificity for HCMV-infected cells in vitro In the presence of HCMV infection, the engineered antibody was able to activate T cells with no HCMV specificity for cytokine production, proliferation, and the expression of phenotype markers unique to T cell activation. These results suggested the potential of engineered bispecific antibodies, such as the construct described here, as prophylactic or therapeutic agents against HCMV reactivation and infection.

Impact of Antibodies and Strain Polymorphisms on Cytomegalovirus Entry and Spread in Fibroblasts and Epithelial Cells.

Cytomegalovirus (CMV) entry into fibroblasts differs from entry into epithelial cells. CMV also spreads cell to cell and can induce syncytia. To gain insights into these processes, 27 antibodies targeting epitopes in CMV virion glycoprotein complexes, including glycoprotein B (gB), gH/gL, and the pentamer, were evaluated for their effects on viral entry and spread. No antibodies inhibited CMV spread in fibroblasts, including those with potent neutralizing activity against fibroblast entry, while all antibodies that neutralized epithelial cell entry also inhibited spread in epithelial cells and a correlation existed between the potencies of these two activities. This suggests that exposure of virions to the cell culture medium is obligatory during spread in epithelial cells but not in fibroblasts. In fibroblasts, the formation of syncytiumlike structures was impaired not only by antibodies to gB or gH/gL but also by antibodies to the pentamer, suggesting a potential role for the pentamer in promoting fibroblast fusion. Four antibodies reacted with linear epitopes near the N terminus of gH, exhibited strain specificity, and neutralized both epithelial cell and fibroblast entry. Five other antibodies recognized conformational epitopes in gH/gL and neutralized both fibroblast and epithelial cell entry. That these antibodies were strain specific for neutralizing fibroblast but not epithelial cell entry suggests that polymorphisms external to certain gH/gL epitopes may influence antibody neutralization during fibroblast but not epithelial cell entry. These findings may have implications for elucidating the mechanisms of CMV entry, spread, and antibody evasion and may assist in determining which antibodies may be most efficacious following active immunization or passive administration.IMPORTANCE Cytomegalovirus (CMV) is a significant cause of birth defects among newborns infected in utero and morbidity and mortality in transplant and AIDS patients. Monoclonal antibodies and vaccines targeting humoral responses are under development for prophylactic or therapeutic use. The findings reported here (i) confirm that cell-to-cell spread of CMV is sensitive to antibody inhibition in epithelial cells but not fibroblasts, (ii) demonstrate that antibodies can restrict the formation in vitro of syncytiumlike structures that resemble syncytial cytomegalic cells that are associated with CMV disease in vivo, and (iii) reveal that neutralization of CMV by antibodies to certain epitopes in gH or gH/gL is both strain and cell type dependent and can be governed by polymorphisms in sequences external to the epitopes. These findings serve to elucidate the mechanisms of CMV entry, spread, and antibody evasion and may have important implications for the development of CMV vaccines and immunotherapeutics.

Neutralization of Diverse Human Cytomegalovirus Strains Conferred by Antibodies Targeting Viral gH/gL/pUL128-131 Pentameric Complex.

Human cytomegalovirus (HCMV) is the leading cause of congenital viral infection, and developing a prophylactic vaccine is of high priority to public health. We recently reported a replication-defective human cytomegalovirus with restored pentameric complex glycoprotein H (gH)/gL/pUL128-131 for prevention of congenital HCMV infection. While the quantity of vaccine-induced antibody responses can be measured in a viral neutralization assay, assessing the quality of such responses, including the ability of vaccine-induced antibodies to cross-neutralize the field strains of HCMV, remains a challenge. In this study, with a panel of neutralizing antibodies from three healthy human donors with natural HCMV infection or a vaccinated animal, we mapped eight sites on the dominant virus-neutralizing antigen-the pentameric complex of glycoprotein H (gH), gL, and pUL128, pUL130, and pUL131. By evaluating the site-specific antibodies in vaccine immune sera, we demonstrated that vaccination elicited functional antiviral antibodies to multiple neutralizing sites in rhesus macaques, with quality attributes comparable to those of CMV hyperimmune globulin. Furthermore, these immune sera showed antiviral activities against a panel of genetically distinct HCMV clinical isolates. These results highlighted the importance of understanding the quality of vaccine-induced antibody responses, which includes not only the neutralizing potency in key cell types but also the ability to protect against the genetically diverse field strains.IMPORTANCE HCMV is the leading cause of congenital viral infection, and development of a preventive vaccine is a high public health priority. To understand the strain coverage of vaccine-induced immune responses in comparison with natural immunity, we used a panel of broadly neutralizing antibodies to identify the immunogenic sites of a dominant viral antigen-the pentameric complex. We further demonstrated that following vaccination of a replication-defective virus with the restored pentameric complex, rhesus macaques can develop broadly neutralizing antibodies targeting multiple immunogenic sites of the pentameric complex. Such analyses of site-specific antibody responses are imperative to our assessment of the quality of vaccine-induced immunity in clinical studies.

Maternal Antibody Responses and Nonprimary Congenital Cytomegalovirus Infection of HIV-1-Exposed Infants.

Risk of congenital cytomegalovirus (cCMV) transmission is highly dependent on the presence of preexisting maternal immunity, with the lowest rates observed in CMV-seroimmune populations. Among infants of CMV-seroimmune women, those who are exposed to human immunodeficiency virus (HIV) have an increased risk of acquiring cCMV infection as compared to HIV-unexposed infants. To better understand the risk factors of nonprimary cCMV transmission in HIV-infected women, we performed a case-control study in which CMV-specific plasma antibody responses from 19 CMV-transmitting and 57 CMV-nontransmitting women with chronic CMV/HIV coinfection were evaluated for the ability to predict the risk of cCMV infection. Primary multivariable conditional logistic regression analysis revealed an association between epithelial-tropic CMV neutralizing titers and a reduced risk of cCMV transmission (odds ratio [OR], 0.18; 95% confidence interval [CI], .03-.93; P = .04), although this effect was not significant following correction for multiple comparisons (false-discovery rate, 0.12). Exploratory analysis of the CMV specificity of plasma antibodies revealed that immunoglobulin G (IgG) responses against the glycoprotein B (gB) neutralizing epitope AD-2 had a borderline association with low risk of transmission (OR, 0.72; 95% CI, .51-1.00; P = .05), although this was not confirmed in a post hoc plasma anti-AD-2 IgG blocking assay. Our data suggest that maternal neutralizing antibody responses may play a role in protection against cCMV in HIV/CMV-coinfected populations.

Soluble Human Cytomegalovirus gH/gL/pUL128-131 Pentameric Complex, but Not gH/gL, Inhibits Viral Entry to Epithelial Cells and Presents Dominant Native Neutralizing Epitopes.

Congenital infection of human cytomegalovirus (HCMV) is one of the leading causes of nongenetic birth defects, and development of a prophylactic vaccine against HCMV is of high priority for public health. The gH/gL/pUL128-131 pentameric complex mediates HCMV entry into endothelial and epithelial cells, and it is a major target for neutralizing antibody responses. To better understand the mechanism by which antibodies interact with the epitopes of the gH/gL/pUL128-131 pentameric complex resulting in viral neutralization, we expressed and purified soluble gH/gL/pUL128-131 pentameric complex and gH/gL from Chinese hamster ovary cells to >95% purity. The soluble gH/gL, which exists predominantly as (gH/gL)2 homodimer with a molecular mass of 220 kDa in solution, has a stoichiometry of 1:1 and a pI of 6.0-6.5. The pentameric complex has a molecular mass of 160 kDa, a stoichiometry of 1:1:1:1:1, and a pI of 7.4-8.1. The soluble pentameric complex, but not gH/gL, adsorbs 76% of neutralizing activities in HCMV human hyperimmune globulin, consistent with earlier reports that the most potent neutralizing epitopes for blocking epithelial infection are unique to the pentameric complex. Functionally, the soluble pentameric complex, but not gH/gL, blocks viral entry to epithelial cells in culture. Our results highlight the importance of the gH/gL/pUL128-131 pentameric complex in HCMV vaccine design and emphasize the necessity to monitor the integrity of the pentameric complex during the vaccine manufacturing process.

Functional analysis of human cytomegalovirus UL/b' region using SCID-hu mouse model.

Human cytomegalovirus (HCMV) attenuated strains, Towne, and AD169, differ from prototypic pathogenic strains, such as Toledo, in that they are missing a ∼15-kb segment in the UL/b' region. In contrast to the attenuated strains, Toledo can replicate in human tissue implants in SCID (SCID-hu) mice. Thus, this model provides a unique in vivo system to study the mechanism of viral pathogenesis. Twenty-two ORFs have been annotated in the UL/b' region, including tissue-tropic genes encoded in a pentameric gH/gl complex. To differentiate the role of the pentameric gH/gl complex versus the functions of other ORFs in the 15-kb region in supporting viral growth in vivo, a series of recombinant viral strains were constructed and their ability to replicate in SCID-hu mice was tested. The mutations in the Towne and AD169 strains were repaired to restore their pentameric gH/gl complex and it was found that these changes did not rescue their inability to replicate in the SCID-hu mice. Subsequently four deletion viruses (D1, D2, D3, and D4) in the 15-kb region from the Toledo strain were created. It was demonstrated that D2 and D3 were able to grow in SCID-hu mice, while D1 and D4 were not viable. Interestingly, co-infection of the implant with the D1 and D4 viruses could compensate their respective growth defect in vivo. The results demonstrated that rescuing viral epithelial tropism is not sufficient to revert the attenuation phenotype of AD169 or Towne, and pathogenic genes are located in the segments missing in D1 and D4 viruses. J. Med. Virol. 88:1417-1426, 2016. © 2016 Wiley Periodicals, Inc.

Pentameric complex of viral glycoprotein H is the primary target for potent neutralization by a human cytomegalovirus vaccine.

Human cytomegalovirus (HCMV) can cause serious morbidity/mortality in transplant patients, and congenital HCMV infection can lead to birth defects. Developing an effective HCMV vaccine is a high medical priority. One of the challenges to the efforts has been our limited understanding of the viral antigens important for protective antibodies. Receptor-mediated viral entry to endothelial/epithelial cells requires a glycoprotein H (gH) complex comprising five viral proteins (gH, gL, UL128, UL130, and UL131). This gH complex is notably missing from HCMV laboratory strains as well as HCMV vaccines previously evaluated in the clinic. To support a unique vaccine concept based on the pentameric gH complex, we established a panel of 45 monoclonal antibodies (mAbs) from a rabbit immunized with an experimental vaccine virus in which the expression of the pentameric gH complex was restored. Over one-half (25 of 45) of the mAbs have neutralizing activity. Interestingly, affinity for an antibody to bind virions was not correlated with its ability to neutralize the virus. Genetic analysis of the 45 mAbs based on their heavy- and light-chain sequences identified at least 26 B-cell linage groups characterized by distinct binding or neutralizing properties. Moreover, neutralizing antibodies possessed longer complementarity-determining region 3 for both heavy and light chains than those with no neutralizing activity. Importantly, potent neutralizing mAbs reacted to the pentameric gH complex but not to gB. Thus, the pentameric gH complex is the primary target for antiviral antibodies by vaccination.

Inhibition of human cytomegalovirus entry into mucosal epithelial cells.

Human cytomegalovirus (CMV) causes serious developmental disabilities in newborns infected in utero following oral acquisition by the mother. Thus, neutralizing antibodies in maternal saliva have potential to prevent maternal infection and, consequently, fetal transmission and disease. Based on standard cell culture models, CMV entry mediators (and hence neutralizing targets) are cell type-dependent: entry into fibroblasts requires glycoprotein B (gB) and a trimeric complex (TC) of glycoproteins H, L, and O, whereas endothelial and epithelial cell entry additionally requires a pentameric complex (PC) of glycoproteins H and L with UL128, UL130, and UL131A. However, as the mediators of mucosal cell entry and the potential impact of cellular differentiation remained unclear, the present studies utilized mutant viruses, neutralizing antibodies, and soluble TC-receptor to determine the entry mediators required for infection of mucocutaneus cell lines and primary tonsil epithelial cells. Entry into undifferentiated cells was largely PC-dependent, but PC-independent entry could be induced by differentiation. TC-independent entry was also observed and varied by cell line and differentiation. Infection of primary tonsil cells from some donors was entirely TC-independent. In contrast, an antibody to gB or disruption of virion attachment using heparin blocked entry into all cells. These findings indicate that CMV entry into the spectrum of cell types encountered in vivo is likely to be more complex than has been suggested by standard cell culture models and may be influenced by the relative abundance of virion envelope glycoprotein complexes as well as by cell type, tissue of origin, and state of differentiation.

SARS-CoV-2 Omicron: Viral Evolution, Immune Evasion, and Alternative Durable Therapeutic Strategies.

Since the SARS-CoV-2 Omicron virus has gained dominance worldwide, its continual evolution with unpredictable mutations and patterns has revoked all authorized immunotherapeutics. Rapid viral evolution has also necessitated several rounds of vaccine updates in order to provide adequate immune protection. It remains imperative to understand how Omicron evolves into different subvariants and causes immune escape as this could help reevaluate the current intervention strategies mostly implemented in the clinics as emergency measures to counter the pandemic and, importantly, develop new solutions. Here, we provide a review focusing on the major events of Omicron viral evolution, including the features of spike mutation that lead to immune evasion against monoclonal antibody (mAb) therapy and vaccination, and suggest alternative durable options such as the ACE2-based experimental therapies superior to mAbs to address this unprecedented evolution of Omicron virus. In addition, this type of unique ACE2-based virus-trapping molecules can counter all zoonotic SARS coronaviruses, either from unknown animal hosts or from established wild-life reservoirs of SARS-CoV-2, and even seasonal alpha coronavirus NL63 that depends on human ACE2 for infection.

Transcriptional signature of durable effector T cells elicited by a replication defective HCMV vaccine.

Human cytomegalovirus (HCMV) is a leading infectious cause of birth defects and the most common opportunistic infection that causes life-threatening diseases post-transplantation; however, an effective vaccine remains elusive. V160 is a live-attenuated replication defective HCMV vaccine that showed a 42.4% efficacy against primary HCMV infection among seronegative women in a phase 2b clinical trial. Here, we integrated the multicolor flow cytometry, longitudinal T cell receptor (TCR) sequencing, and single-cell RNA/TCR sequencing approaches to characterize the magnitude, phenotype, and functional quality of human T cell responses to V160. We demonstrated that V160 de novo induces IE-1 and pp65 specific durable polyfunctional effector CD8 T cells that are comparable to those induced by natural HCMV infection. We identified a variety of V160-responsive T cell clones which exhibit distinctive "transient" and "durable" expansion kinetics, and revealed a transcriptional signature that marks durable CD8 T cells post-vaccination. Our study enhances the understanding of human T-cell immune responses to V160 vaccination.

An ACE2 decamer viral trap as a durable intervention solution for current and future SARS-CoV.

The capacity of SARS-CoV-2 to evolve poses challenges to conventional prevention and treatment options such as vaccination and monoclonal antibodies, as they rely on viral receptor binding domain (RBD) sequences from previous strains. Additionally, animal CoVs, especially those of the SARS family, are now appreciated as a constant pandemic threat. We present here a new antiviral approach featuring inhalation delivery of a recombinant viral trap composed of ten copies of angiotensin-converting enzyme 2 (ACE2) fused to the IgM Fc. This ACE2 decamer viral trap is designed to inhibit SARS-CoV-2 entry function, regardless of viral RBD sequence variations as shown by its high neutralization potency against all known SARS-CoV-2 variants, including Omicron BQ.1, BQ.1.1, XBB.1 and XBB.1.5. In addition, it demonstrates potency against SARS-CoV-1, human NL63, as well as bat and pangolin CoVs. The multivalent trap is effective in both prophylactic and therapeutic settings since a single intranasal dosing confers protection in human ACE2 transgenic mice against viral challenges. Lastly, this molecule is stable at ambient temperature for more than twelve weeks and can sustain physical stress from aerosolization. These results demonstrate the potential of a decameric ACE2 viral trap as an inhalation solution for ACE2-dependent coronaviruses of current and future pandemic concerns.

Evidence for cyclic diguanylate as a vaccine adjuvant with novel immunostimulatory activities.

Cyclic diguanylate (c-di-GMP), a bacterial signaling molecule, possesses protective immunostimulatory activity in bacterial challenge models. This study explored the potential of c-di-GMP as a vaccine adjuvant comparing it with LPS, CpG oligonucleotides, and a conventional aluminum salt based adjuvant. In this evaluation, c-di-GMP was a more potent activator of both humoral and Th1-like immune responses as evidenced by the robust IgG2a antibody response it induced in mice and the strong IFN-γ, TNF-α and IP-10 responses, it elicited in mice and in vitro in non-human primate peripheral blood mononuclear cells. Further, compared to LPS or CpG, c-di-GMP demonstrated a more pronounced ability to induce germinal center formation, a hallmark of long-term memory, in immunized mice. Together, these data add to the growing body of evidence supporting the utility of c-di-GMP as an adjuvant in vaccination for sustained and robust immune responses and provide a rationale for further evaluation in appropriate models of immunization.