Mucosal boosting enhances vaccine protection against SARS-CoV-2 in macaques.

A limitation of current SARS-CoV-2 vaccines is that they provide minimal protection against infection with current Omicron subvariants1,2, although they still provide protection against severe disease. Enhanced mucosal immunity may be required to block infection and onward transmission. Intranasal administration of current vaccines has proven inconsistent3-7, suggesting that alternative immunization strategies may be required. Here we show that intratracheal boosting with a bivalent Ad26-based SARS-CoV-2 vaccine results in substantial induction of mucosal humoral and cellular immunity and near-complete protection against SARS-CoV-2 BQ.1.1 challenge. A total of 40 previously immunized rhesus macaques were boosted with a bivalent Ad26 vaccine by the intramuscular, intranasal and intratracheal routes, or with a bivalent mRNA vaccine by the intranasal route. Ad26 boosting by the intratracheal route led to a substantial expansion of mucosal neutralizing antibodies, IgG and IgA binding antibodies, and CD8+ and CD4+ T cell responses, which exceeded those induced by Ad26 boosting by the intramuscular and intranasal routes. Intratracheal Ad26 boosting also led to robust upregulation of cytokine, natural killer, and T and B cell pathways in the lungs. After challenge with a high dose of SARS-CoV-2 BQ.1.1, intratracheal Ad26 boosting provided near-complete protection, whereas the other boosting strategies proved less effective. Protective efficacy correlated best with mucosal humoral and cellular immune responses. These data demonstrate that these immunization strategies induce robust mucosal immunity, suggesting the feasibility of developing vaccines that block respiratory viral infections.

Mechanistic Model Describing the Time Course of Humoral Immunity Following Ad26.COV2.S Vaccination in Non-Human Primates.

Mechanistic modeling can be used to describe the time course of vaccine-induced humoral immunity and to identify key biologic drivers in antibody production. We used a six-compartment mechanistic model to describe a 20-week time course of humoral immune responses in 56 non-human primates (NHPs) elicited by vaccination with Ad26.COV2.S according to either a single-dose regimen (1 × 1011 or 5 × 1010 viral particles [vp]) or a two-dose homologous regimen (5 × 1010 vp) given in an interval of 4 or 8 weeks. Humoral immune responses were quantified by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike-specific binding antibody concentrations as determined by spike protein-enzyme-linked immunosorbent assay. The mechanistic model adequately described the central tendency and variability of binding antibody concentrations through 20 weeks in all vaccination arms. The estimation of mechanistic modeling parameters revealed greater contribution of the antibody production mediated by short-lived cells as compared with long-lived cells in driving the peak response, especially post second dose when a more rapid peak response was observed. The antibody production mediated by long-lived cells was identified as relevant for generating the first peak and for contributing to the long-term time course of sustained antibody concentrations in all vaccination arms. The findings contribute evidence on the key biologic components responsible for the observed time course of vaccine-induced humoral immunity in NHPs and constitute a step toward defining immune biomarkers of protection against SARS-CoV-2 that might translate across species. SIGNIFICANCE STATEMENT: We demonstrate the adequacy of a mechanistic modeling approach describing the time course of binding antibody concentrations in non-human primates (NHPs) elicited by different dose levels and regimens of Ad26.COV2.S. The findings are relevant for informing the mechanism-based accounts of vaccine-induced humoral immunity in NHPs and translational research efforts aimed at identifying immune biomarkers of protection against SARS-CoV-2 infection.

Dynamics of the Humoral Immune Response to a Prime-Boost Ebola Vaccine: Quantification and Sources of Variation.

The Ebola vaccine based on Ad26.ZEBOV/MVA-BN-Filo prime-boost regimens is being evaluated in multiple clinical trials. The long-term immune response to the vaccine is unknown, including factors associated with the response and variability around the response. We analyzed data from three phase 1 trials performed by the EBOVAC1 Consortium in four countries: the United Kingdom, Kenya, Tanzania, and Uganda. Participants were randomized into four groups based on the interval between prime and boost immunizations (28 or 56 days) and the sequence in which Ad26.ZEBOV and MVA-BN-Filo were administered. Consecutive enzyme-linked immunosorbent assay (ELISA) measurements of the IgG binding antibody concentrations against the Kikwit glycoprotein (GP) were available for 177 participants to assess the humoral immune response up to 1 year postprime. Using a mathematical model for the dynamics of the humoral response, from 7 days after the boost immunization up to 1 year after the prime immunization, we estimated the durability of the antibody response and the influence of different factors on the dynamics of the humoral response. Ordinary differential equations (ODEs) described the dynamics of antibody response and two populations of antibody-secreting cells (ASCs), short-lived (SL) and long-lived (LL). Parameters of the ODEs were estimated using a population approach. We estimated that half of the LL ASCs could persist for at least 5 years. The vaccine regimen significantly affected the SL ASCs and the antibody peak but not the long-term response. The LL ASC compartment dynamics differed significantly by geographic regions analyzed, with a higher long-term antibody persistence in European subjects. These differences could not be explained by the observed differences in cellular immune response.IMPORTANCE With no available licensed vaccines or therapies, the West African Ebola virus disease epidemic of 2014 to 2016 caused 11,310 deaths. Following this outbreak, the development of vaccines has been accelerated. Combining different vector-based vaccines as heterologous regimens could induce a durable immune response, assessed through antibody concentrations. Based on data from phase 1 trials in East Africa and Europe, the dynamics of the humoral immune response from 7 days after the boost immunization onwards were modeled to estimate the durability of the response and understand its variability. Antibody production is maintained by a population of long-lived cells. Estimation suggests that half of these cells can persist for at least 5 years in humans. Differences in prime-boost vaccine regimens affect only the short-term immune response. Geographical differences in long-lived cell dynamics were inferred, with higher long-term antibody concentrations induced in European participants.

A model for establishment, maintenance and reactivation of the immune response after vaccination against Ebola virus.

The 2014-2016 Ebola outbreak in West Africa has triggered accelerated development of several preventive vaccines against Ebola virus. Under the EBOVAC1 consortium, three phase I studies were carried out to assess safety and immunogenicity of a two-dose heterologous vaccination regimen developed by Janssen Vaccines and Prevention in collaboration with Bavarian Nordic. To describe the immune response induced by the two-dose heterologous vaccine regimen, we propose a mechanistic ODE based model, which takes into account the role of immunological memory. We perform identifiability and sensitivity analysis of the proposed model to establish which kind of biological data are ideally needed in order to accurately estimate parameters, and additionally, which of those are non-identifiable based on the available data. Antibody concentrations data from phase I studies have been used to calibrate the model and show its ability in reproducing the observed antibody dynamics. Together with other factors, the establishment of an effective and reactive immunological memory is of pivotal importance for several prophylactic vaccines. We show that introducing a memory compartment in our calibrated model allows to evaluate the magnitude of the immune response induced by a booster dose and its long-term persistence afterwards.

Lack of prion infectivity in fixed heart tissue from patients with Creutzfeldt-Jakob disease or amyloid heart disease.

In most forms of prion disease, infectivity is present primarily in the central nervous system or immune system organs such as spleen and lymph node. However, a transgenic mouse model of prion disease has demonstrated that prion infectivity can also be present as amyloid deposits in heart tissue. Deposition of infectious prions as amyloid in human heart tissue would be a significant public health concern. Although abnormal disease-associated prion protein (PrP(Sc)) has not been detected in heart tissue from several amyloid heart disease patients, it has been observed in the heart tissue of a patient with sporadic Creutzfeldt-Jakob Disease (sCJD), the most common form of human prion disease. In order to determine whether prion infectivity can be found in heart tissue, we have inoculated formaldehyde fixed brain and heart tissue from two sCJD patients, as well as prion protein positive fixed heart tissue from two amyloid heart disease patients, into transgenic mice overexpressing the human prion protein. Although the sCJD brain samples led to clinical or subclinical prion infection and deposition of PrP(Sc) in the brain, none of the inoculated heart samples resulted in disease or the accumulation of PrP(Sc). Thus, our results suggest that prion infectivity is not likely present in cardiac tissue from sCJD or amyloid heart disease patients.

The Biodistribution of the Spike Protein after Ad26.COV2.S Vaccination Is Unlikely to Play a Role in Vaccine-Induced Immune Thrombotic Thrombocytopenia.

Ad26.COV2.S vaccination can lead to vaccine-induced immune thrombotic thrombocytopenia (VITT), a rare but severe adverse effect, characterized by thrombocytopenia and thrombosis. The mechanism of VITT induction is unclear and likely multifactorial, potentially including the activation of platelets and endothelial cells mediated by the vaccine-encoded spike protein (S protein). Here, we investigated the biodistribution of the S protein after Ad26.COV2.S dosing in three animal models and in human serum samples. The S protein was transiently present in draining lymph nodes of rabbits after Ad26.COV2.S dosing. The S protein was detected in the serum in all species from 1 day to 21 days after vaccination with Ad26.COV2.S, but it was not detected in platelets, the endothelium lining the blood vessels, or other organs. The S protein S1 and S2 subunits were detected at different ratios and magnitudes after Ad26.COV2.S or COVID-19 mRNA vaccine immunization. However, the S1/S2 ratio did not depend on the Ad26 platform, but on mutation of the furin cleavage site, suggesting that the S1/S2 ratio is not VITT related. Overall, our data suggest that the S-protein biodistribution and kinetics after Ad26.COV2.S dosing are likely not main contributors to the development of VITT, but other S-protein-specific parameters require further investigation.

JC polyomavirus (JCV) and monoclonal antibodies: friends or potential foes?

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system (CNS), observed in immunodeficient patients and caused by JC virus ((JCV), also called JC polyomavirus (JCPyV)). After the HIV pandemic and the introduction of immunomodulatory therapy, the PML incidence significantly increased. The correlation between the use of natalizumab, a drug used in multiple sclerosis (MS), and the PML development of particular relevance. The high incidence of PML in natalizumab-treated patients has highlighted the importance of two factors: the need of PML risk stratification among natalizumab-treated patients and the need of effective therapeutic options. In this review, we discuss these two needs under the light of the major viral models of PML etiopathogenesis.

Persistence of immunological memory as a potential correlate of long-term, vaccine-induced protection against Ebola virus disease in humans.

Introduction: In the absence of clinical efficacy data, vaccine protective effect can be extrapolated from animals to humans, using an immunological biomarker in humans that correlates with protection in animals, in a statistical approach called immunobridging. Such an immunobridging approach was previously used to infer the likely protective effect of the heterologous two-dose Ad26.ZEBOV, MVA-BN-Filo Ebola vaccine regimen. However, this immunobridging model does not provide information on how the persistence of the vaccine-induced immune response relates to durability of protection in humans. Methods and results: In both humans and non-human primates, vaccine-induced circulating antibody levels appear to be very stable after an initial phase of contraction and are maintained for at least 3.8 years in humans (and at least 1.3 years in non-human primates). Immunological memory was also maintained over this period, as shown by the kinetics and magnitude of the anamnestic response following re-exposure to the Ebola virus glycoprotein antigen via booster vaccination with Ad26.ZEBOV in humans. In non-human primates, immunological memory was also formed as shown by an anamnestic response after high-dose, intramuscular injection with Ebola virus, but was not sufficient for protection against Ebola virus disease at later timepoints due to a decline in circulating antibodies and the fast kinetics of disease in the non-human primates model. Booster vaccination within three days of subsequent Ebola virus challenge in non-human primates resulted in protection from Ebola virus disease, i.e. before the anamnestic response was fully developed. Discussion: Humans infected with Ebola virus may benefit from the anamnestic response to prevent disease progression, as the incubation time is longer and progression of Ebola virus disease is slower as compared to non-human primates. Therefore, the persistence of vaccine-induced immune memory could be considered as a potential correlate of long-term protection against Ebola virus disease in humans, without the need for a booster.

Booster with Ad26.COV2.S or Omicron-adapted vaccine enhanced immunity and efficacy against SARS-CoV-2 Omicron in macaques.

Omicron spike (S) encoding vaccines as boosters, are a potential strategy to improve COVID-19 vaccine efficacy against Omicron. Here, macaques (mostly females) previously immunized with Ad26.COV2.S, are boosted with Ad26.COV2.S, Ad26.COV2.S.529 (encoding Omicron BA.1 S) or a 1:1 combination of both vaccines. All booster vaccinations elicit a rapid antibody titers increase against WA1/2020 and Omicron S. Omicron BA.1 and BA.2 antibody responses are most effectively boosted by vaccines including Ad26.COV2.S.529. Independent of vaccine used, mostly WA1/2020-reactive or WA1/2020-Omicron BA.1 cross-reactive B cells are detected. Ad26.COV2.S.529 containing boosters provide only slightly higher protection of the lower respiratory tract against Omicron BA.1 challenge compared with Ad26.COV2.S-only booster. Antibodies and cellular immune responses are identified as complementary correlates of protection. Overall, a booster with an Omicron-spike based vaccine provide only moderately improved immune responses and protection compared with the original Wuhan-Hu-1-spike based vaccine, which still provide robust immune responses and protection against Omicron.

HCV proteins and immunoglobulin variable gene (IgV) subfamilies in HCV-induced type II mixed cryoglobulinemia: a concurrent pathogenetic role.

The association between hepatitis C virus (HCV) infection and type II mixed cryoglobulinemia (MCII) is well established, but the role played by distinct HCV proteins and by specific components of the anti-HCV humoral immune response remains to be clearly defined. It is widely accepted that HCV drives the expansion of few B-cell clones expressing a restricted pool of selected immunoglobulin variable (IgV) gene subfamilies frequently endowed with rheumatoid factor (RF) activity. Moreover, the same IgV subfamilies are frequently observed in HCV-transformed malignant B-cell clones occasionally complicating MCII. In this paper, we analyze both the humoral and viral counterparts at the basis of cryoglobulins production in HCV-induced MCII, with particular attention reserved to the single IgV subfamilies most frequently involved.

Neutralization activity and kinetics of two broad-range human monoclonal IgG1 derived from recombinant Fab fragments and directed against Hepatitis C virus E2 glycoprotein.

Hepatitis C virus (HCV) is the major cause of chronic liver disease worldwide. There is evidence that neutralizing anti-HCV antibodies may find potential applications in novel prophylactic and therapeutic strategies. This paper describes the very high neutralization activity and unique biological features of two broadly cross-reactive and cross-neutralizing anti-HCV human monoclonal IgG1 derived from human monoclonal recombinant Fab fragments.

A phage display vector optimized for the generation of human antibody combinatorial libraries and the molecular cloning of monoclonal antibody fragments.

A novel phagemid vector, named pCM, was optimized for the cloning and display of antibody fragment (Fab) libraries on the surface of filamentous phage. This vector contains two long DNA "stuffer" fragments for easier differentiation of the correctly cut forms of the vector. Moreover, in pCM the fragment at the heavy-chain cloning site contains an acid phosphatase-encoding gene allowing an easy distinction of the Escherichia coli cells containing the unmodified form of the phagemid versus the heavy-chain fragment coding cDNA. In pCM transcription of heavy-chain Fd/gene III and light chain is driven by a single lacZ promoter. The light chain is directed to the periplasm by the ompA signal peptide, whereas the heavy-chain Fd/coat protein III is trafficked by the pelB signal peptide. The phagemid pCM was used to generate a human combinatorial phage display antibody library that allowed the selection of a monoclonal Fab fragment antibody directed against the nucleoprotein (NP) of Influenza A virus.

Phage display-based strategies for cloning and optimization of monoclonal antibodies directed against human pathogens.

In the last two decades, several phage display-selected monoclonal antibodies (mAbs) have been described in the literature and a few of them have managed to reach the clinics. Among these, the anti-respiratory syncytial virus (RSV) Palivizumab, a phage-display optimized mAb, is the only marketed mAb directed against microbial pathogens. Palivizumab is a clear example of the importance of choosing the most appropriate strategy when selecting or optimizing an anti-infectious mAb. From this perspective, the extreme versatility of phage-display technology makes it a useful tool when setting up different strategies for the selection of mAbs directed against human pathogens, especially when their possible clinical use is considered. In this paper, we review the principal phage display strategies used to select anti-infectious mAbs, with particular attention focused on those used against hypervariable pathogens, such as HCV and influenza viruses.

Protection against Marburg Virus and Sudan Virus in NHP by an Adenovector-Based Trivalent Vaccine Regimen Is Correlated to Humoral Immune Response Levels.

The Marburg virus (MARV) and Sudan virus (SUDV) belong to the filovirus family. The sporadic human outbreaks occur mostly in Africa and are characterized by an aggressive disease course with high mortality. The first case of Marburg virus disease in Guinea in 2021, together with the increased frequency of outbreaks of Ebola virus (EBOV), which is also a filovirus, accelerated the interest in potential prophylactic vaccine solutions against multiple filoviruses. We previously tested a two-dose heterologous vaccine regimen (Ad26.Filo, MVA-BN-Filo) in non-human primates (NHP) and showed a fully protective immune response against both SUDV and MARV in addition to the already-reported protective effect against EBOV. The vaccine-induced glycoprotein (GP)-binding antibody levels appear to be good predictors of the NHP challenge outcome as indicated by the correlation between antibody levels and survival outcome as well as the high discriminatory capacity of the logistic model. Moreover, the elicited GP-specific binding antibody response against EBOV, SUDV, and MARV remains stable for more than 1 year. Overall, the NHP data indicate that the Ad26.Filo, MVA-BN-Filo regimen may be a good candidate for a prophylactic vaccination strategy in regions at high risk of filovirus outbreaks.

Non-human primate to human immunobridging demonstrates a protective effect of Ad26.ZEBOV, MVA-BN-Filo vaccine against Ebola.

Without clinical efficacy data, vaccine protective effect may be extrapolated from animals to humans using an immunologic marker that correlates with protection in animals. This immunobridging approach was used for the two-dose Ebola vaccine regimen Ad26.ZEBOV, MVA-BN-Filo. Ebola virus (EBOV) glycoprotein binding antibody data obtained from 764 vaccinated healthy adults in five clinical studies (NCT02416453, NCT02564523, NCT02509494, NCT02543567, NCT02543268) were used to calculate mean predicted survival probability (with preplanned 95% confidence interval [CI]). We used a logistic regression model based on EBOV glycoprotein binding antibody responses in vaccinated non-human primates (NHPs) and NHP survival after EBOV challenge. While the protective effect of the vaccine regimen in humans can be inferred in this fashion, the extrapolated survival probability cannot be directly translated into vaccine efficacy. The primary immunobridging analysis evaluated the lower limit of the CI against predefined success criterion of 20% and passed with mean predicted survival probability of 53.4% (95% CI: 36.7-67.4).

Histidines in the octapeptide repeat of PrPC react with PrPSc at an acidic pH.

Cellular PrP is actively cycled between the cell surface and the endosomal pathway. The exact site and mechanism of conversion from PrP(C) to PrP(Sc) remain unknown. We have previously used recombinant antibodies containing grafts of PrP sequence to identify three regions of PrP(C) (aa23-27, 98-110, and 136-158) that react with PrP(Sc) at neutral pH. To determine if any regions of PrP(C) react with PrP(Sc) at an acidic pH similar to that of an endosomal compartment, we tested our panel of grafted antibodies for the ability to precipitate PrP(Sc) in a range of pH conditions. At pH near or lower than 6, PrP-grafted antibodies representing the octapeptide repeat react strongly with PrP(Sc) but not PrP(C). Modified grafts in which the histidines of the octarepeat were replaced with alanines did not react with PrP(Sc). PrP(Sc) precipitated by the octapeptide at pH 5.7 was able to seed conversion of normal PrP to PrP(Sc) in vitro. However, modified PrP containing histidine to alanine substitutions within the octapeptide repeats was still converted to PrP(Sc) in N2a cells. These results suggest that once PrP has entered the endosomal pathway, the acidic environment facilitates the binding of PrP(Sc) to the octarepeat of PrP(C) by the change in charge of the histidines within the octarepeat.

SARS-CoV-2 binding and neutralizing antibody levels after Ad26.COV2.S vaccination predict durable protection in rhesus macaques.

Several COVID-19 vaccines have recently gained authorization for emergency use. Limited knowledge on duration of immunity and efficacy of these vaccines is currently available. Data on other coronaviruses after natural infection suggest that immunity to SARS-CoV-2 might be short-lived, and preliminary evidence indicates waning antibody titers following SARS-CoV-2 infection. In this work, we model the relationship between immunogenicity and protective efficacy of a series of Ad26 vectors encoding stabilized variants of the SARS-CoV-2 Spike protein in rhesus macaques and validate the analyses by challenging macaques 6 months after immunization with the Ad26.COV2.S vaccine candidate that has been selected for clinical development. We show that Ad26.COV2.S confers durable protection against replication of SARS-CoV-2 in the lungs that is predicted by the levels of Spike-binding and neutralizing antibodies, indicating that Ad26.COV2.S could confer durable protection in humans and immunological correlates of protection may enable the prediction of durability of protection.

Immunogenicity and efficacy of one and two doses of Ad26.COV2.S COVID vaccine in adult and aged NHP.

Safe and effective coronavirus disease-19 (COVID-19) vaccines are urgently needed to control the ongoing pandemic. While single-dose vaccine regimens would provide multiple advantages, two doses may improve the magnitude and durability of immunity and protective efficacy. We assessed one- and two-dose regimens of the Ad26.COV2.S vaccine candidate in adult and aged nonhuman primates (NHPs). A two-dose Ad26.COV2.S regimen induced higher peak binding and neutralizing antibody responses compared with a single dose. In one-dose regimens, neutralizing antibody responses were stable for at least 14 wk, providing an early indication of durability. Ad26.COV2.S induced humoral immunity and T helper cell (Th cell) 1-skewed cellular responses in aged NHPs that were comparable to those in adult animals. Aged Ad26.COV2.S-vaccinated animals challenged 3 mo after dose 1 with a SARS-CoV-2 spike G614 variant showed near complete lower and substantial upper respiratory tract protection for both regimens. Neutralization of variants of concern by NHP sera was reduced for B.1.351 lineages while maintained for the B.1.1.7 lineage independent of Ad26.COV2.S vaccine regimen.

Nonhuman primate to human immunobridging to infer the protective effect of an Ebola virus vaccine candidate.

It has been proven challenging to conduct traditional efficacy trials for Ebola virus (EBOV) vaccines. In the absence of efficacy data, immunobridging is an approach to infer the likelihood of a vaccine protective effect, by translating vaccine immunogenicity in humans to a protective effect, using the relationship between vaccine immunogenicity and the desired outcome in a suitable animal model. We here propose to infer the protective effect of the Ad26.ZEBOV, MVA-BN-Filo vaccine regimen with an 8-week interval in humans by immunobridging. Immunogenicity and protective efficacy data were obtained for Ad26.ZEBOV and MVA-BN-Filo vaccine regimens using a fully lethal EBOV Kikwit challenge model in cynomolgus monkeys (nonhuman primates [NHP]). The association between EBOV neutralizing antibodies, glycoprotein (GP)-binding antibodies, and GP-reactive T cells and survival in NHP was assessed by logistic regression analysis. Binding antibodies against the EBOV surface GP were identified as the immune parameter with the strongest correlation to survival post EBOV challenge, and used to infer the predicted protective effect of the vaccine in humans using published data from phase I studies. The human vaccine-elicited EBOV GP-binding antibody levels are in a range associated with significant protection against mortality in NHP. Based on this immunobridging analysis, the EBOV GP-specific-binding antibody levels elicited by the Ad26.ZEBOV, MVA-BN-Filo vaccine regimen in humans will likely provide protection against EBOV disease.