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Therapeutic vaccines that elicit cytotoxic T cell responses targeting tumor-specific neoantigens hold promise for providing long-term clinical benefit to patients with cancer. Here we evaluated safety and tolerability of a therapeutic vaccine encoding 20 shared neoantigens derived from selected common oncogenic driver mutations as primary endpoints in an ongoing phase 1/2 study in patients with advanced/metastatic solid tumors. Secondary endpoints included immunogenicity, overall response rate, progression-free survival and overall survival. Eligible patients were selected if their tumors expressed one of the human leukocyte antigen-matched tumor mutations included in the vaccine, with the majority of patients (18/19) harboring a mutation in KRAS. The vaccine regimen, consisting of a chimp adenovirus (ChAd68) and self-amplifying mRNA (samRNA) in combination with the immune checkpoint inhibitors ipilimumab and nivolumab, was shown to be well tolerated, with observed treatment-related adverse events consistent with acute inflammation expected with viral vector-based vaccines and immune checkpoint blockade, the majority grade 1/2. Two patients experienced grade 3/4 serious treatment-related adverse events that were also dose-limiting toxicities. The overall response rate was 0%, and median progression-free survival and overall survival were 1.9 months and 7.9 months, respectively. T cell responses were biased toward human leukocyte antigen-matched TP53 neoantigens encoded in the vaccine relative to KRAS neoantigens expressed by the patients' tumors, indicating a previously unknown hierarchy of neoantigen immunodominance that may impact the therapeutic efficacy of multiepitope shared neoantigen vaccines. These data led to the development of an optimized vaccine exclusively targeting KRAS-derived neoantigens that is being evaluated in a subset of patients in phase 2 of the clinical study. ClinicalTrials.gov registration: NCT03953235 ., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

SARS-CoV-2 has resulted in high levels of morbidity and mortality world-wide, and severe complications can occur in older populations. Humoral immunity induced by authorized vaccines wanes within 6 months, and frequent boosts may only offer transient protection. GRT-R910 is an investigational self-amplifying mRNA (samRNA)-based SARS-CoV-2 vaccine delivering full-length Spike and selected conserved non-Spike T cell epitopes. This study reports interim analyses for a phase I open-label dose-escalation trial evaluating GRT-R910 in previously vaccinated healthy older adults (NCT05148962). Primary endpoints of safety and tolerability were assessed. Most solicited local and systemic adverse events (AEs) following GRT-R910 dosing were mild to moderate and transient, and no treatment-related serious AEs were observed. The secondary endpoint of immunogenicity was assessed via IgG binding assays, neutralization assays, interferon-gamma ELISpot, and intracellular cytokine staining. Neutralizing antibody titers against ancestral Spike and variants of concern were boosted or induced by GRT-R910 and, contrasting to authorized vaccines, persisted through at least 6 months after the booster dose. GRT-R910 increased and/or broadened functional Spike-specific T cell responses and primed functional T cell responses to conserved non-Spike epitopes. This study is limited due to small sample size, and additional data from ongoing studies will be required to corroborate these interim findings., (© 2023. The Author(s).)

Checkpoint inhibitor (CPI) therapies provide limited benefit to patients with tumors of low immune reactivity. T cell-inducing vaccines hold promise to exert long-lasting disease control in combination with CPI therapy. Safety, tolerability and recommended phase 2 dose (RP2D) of an individualized, heterologous chimpanzee adenovirus (ChAd68) and self-amplifying mRNA (samRNA)-based neoantigen vaccine in combination with nivolumab and ipilimumab were assessed as primary endpoints in an ongoing phase 1/2 study in patients with advanced metastatic solid tumors (NCT03639714). The individualized vaccine regimen was safe and well tolerated, with no dose-limiting toxicities. Treatment-related adverse events (TRAEs) >10% included pyrexia, fatigue, musculoskeletal and injection site pain and diarrhea. Serious TRAEs included one count each of pyrexia, duodenitis, increased transaminases and hyperthyroidism. The RP2D was 10 12 viral particles (VP) ChAd68 and 30 µg samRNA. Secondary endpoints included immunogenicity, feasibility of manufacturing and overall survival (OS). Vaccine manufacturing was feasible, with vaccination inducing long-lasting neoantigen-specific CD8 T cell responses. Several patients with microsatellite-stable colorectal cancer (MSS-CRC) had improved OS. Exploratory biomarker analyses showed decreased circulating tumor DNA (ctDNA) in patients with prolonged OS. Although small study size limits statistical and translational analyses, the increased OS observed in MSS-CRC warrants further exploration in larger randomized studies., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The coronavirus disease 2019 (COVID-19) pandemic continues to spread globally, highlighting the urgent need for safe and effective vaccines that could be rapidly mobilized to immunize large populations. We report the preclinical development of a self-amplifying mRNA (SAM) vaccine encoding a prefusion stabilized severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein and demonstrate strong cellular and humoral immune responses at low doses in mice and rhesus macaques. The homologous prime-boost vaccination regimen of SAM at 3, 10 and 30 μg induced potent neutralizing antibody (nAb) titers in rhesus macaques following two SAM vaccinations at all dose levels, with the 10 μg dose generating geometric mean titers (GMT) 48-fold greater than the GMT of a panel of SARS-CoV-2 convalescent human sera. Spike-specific T cell responses were observed with all tested vaccine regimens. SAM vaccination provided protective efficacy against SARS-CoV-2 challenge as both a homologous prime-boost and as a single boost following ChAd prime, demonstrating reduction of viral replication in both the upper and lower airways. The SAM vaccine is currently being evaluated in clinical trials as both a homologous prime-boost regimen at low doses and as a boost following heterologous prime., (© 2022. The Author(s).)

A vaccine against Respiratory Syncytial Virus (RSV) is a major unmet need to prevent the significant morbidity and mortality that it causes in society. In addition to efficacy, such a vaccine must not induce adverse events, as previously occurred with a formalin-inactivated vaccine (FI-RSV). In this study, the safety, immunogenicity and efficacy of a molecularly adjuvanted adenovirus serotype 5 based RSV vaccine encoding the fusion (F) protein (Ad-RSVF) is demonstrated in cotton rats. Protective immunity to RSV was induced by Ad-RSVF when administered by an oral route as well as by intranasal and intramuscular routes. Compared to FI-RSV, the Ad-RSVF vaccine induced significantly greater neutralizing antibody responses and protection against RSV infection. Significantly, oral or intranasal immunization each induced protective multi-functional effector and memory B cell responses in the respiratory tract. This study uniquely demonstrates the capacity of an orally administered adenovirus vaccine to induce protective immunity in the respiratory tract against RSV in a pre-clinical model and supports further clinical development of this oral Ad-RSVF vaccine strategy., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Introduction: Oral vaccines delivered as tablets offer a number of advantages over traditional parenteral-based vaccines including the ease of delivery, lack of needles, no need for trained medical personnel, and the ability to formulate into temperature-stable tablets. We have been evaluating an oral vaccine platform based on recombinant adenoviral vectors for the purpose of creating a prophylactic vaccine to prevent influenza, and have demonstrated vaccine efficacy in animal models and substantial immunogenicity in humans. These studies have evaluated monovalent vaccines to date. To protect against the major circulating A and B influenza strains, a multivalent influenza vaccine will be required., Methods: In this study, the immunogenicity of orally delivered monovalent, bivalent, trivalent, and quadrivalent vaccines was tested in ferrets and mice. The various vaccine combinations were tested by blending monovalent recombinant adenovirus vaccines, each expressing hemagglutinin from a single strain. Human tablet delivery was modeled in animals by oral gavage in mice and by endoscopic delivery in ferrets., Results: We demonstrated minimal interference between the various vaccine vectors when used in combination and that the oral quadrivalent vaccine compared favorably to an approved trivalent inactivated vaccine., Conclusion: The quadrivalent vaccine presented here produced immune responses that we predict should be capable of providing protection against multiple influenza strains, and the platform should have applications to other multivalent vaccines., Funding: Vaxart, Inc.

Purpose: To test the safety and immunogenicity of an orally delivered avian influenza vaccine. The vaccine has a non-replicating adenovirus type 5 vector backbone which expresses hemagglutinin from avian influenza and a TLR3 ligand as an adjuvant., Methods: Forty-two subjects were randomized into 3 groups dosed with either 1×10(10), 1×10(9), or 1×10(8) IU of the vaccine administered in capsules. Twelve subjects were vaccinated with identical capsules containing placebo. A portion of the 1×10(9) dose group were immunized a second time 4 weeks after the first immunization. The safety of the vaccine was assessed by measuring the frequency and severity of adverse events in placebo versus vaccine treated subjects. IFN-γ and granzyme B ELISpot assays were used to assess immunogenicity., Results: The vaccine had a positive safety profile with no treatment emergent adverse events reported above grade 1, and with an adverse event frequency in the treated groups no greater than placebo. Antigen specific cytotoxic and IFN-γ responses were induced in a dose dependent manner and cytotoxic responses were boosted after a second vaccination., Conclusion: This first in man clinical trial demonstrates that an orally delivered adenovirus vectored vaccine can induce immune responses to antigen with a favorable safety profile., Clinical Trial Registration Number: NCT01335347., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

An oral gene-based avian influenza vaccine would allow rapid development and simplified distribution, but efficacy has previously been difficult to achieve by the oral route. This study assessed protection against avian influenza virus challenge using a chimeric adenovirus vector expressing hemagglutinin and a double-stranded RNA adjuvant. Immunized ferrets and mice were protected upon lethal challenge. Further, ferrets immunized by the peroral route induced cross-clade neutralizing antibodies, and the antibodies were selective against hemagglutinin, not the vector. Similarly, experiments in mice demonstrated selective immune responses against HA with peroral delivery and the ability to circumvent preexisting vector immunity.

Adenoviral vaccines delivered orally have been used for decades to prevent respiratory illness, but are now being seriously explored again as a platform technology to make vaccines against a variety of pathogens. Years of use in military populations as a preventative measure for adenoviral infection have demonstrated the safety of oral administration of adenovirus. The advantages of using this approach as a platform technology for vaccines include rapid development and distribution, as well as ease of administration. Recent discoveries may allow this platform approach to reach the clinic within a few years.

In a clinical study of recombinant adeno-associated virus-2 expressing human factor IX (AAV2-FIX), we detected 2 impediments to long-term gene transfer. First, preexisting anti-AAV neutralizing antibodies (NABs) prevent vector from reaching the target tissue, and second, CD8(+) T-cell responses to hepatocyte-cell surface displayed AAV-capsid-terminated FIX expression after several weeks. Because the vector is incapable of synthesizing viral proteins, a short course of immunosuppression, until AAV capsid is cleared from the transduced cells, may mitigate the host T-cell response, allowing long-term expression of FIX. To evaluate coad-ministration of immunosuppression, we studied AAV8 vector infusion in rhesus macaques, natural hosts for AAV8. We administered AAV8-FIX in 16 macaques via the hepatic artery and assessed the effects of (1) preexisting anti-AAV8 NABs, (2) a standard T-cell immunosuppressive regimen, and (3) efficacy and safety of AAV8-FIX. We found that low titers (1:5) of preexisting NABs abrogate transduction, whereas animals with undetectable NABs are safely and effectively transduced by AAV8-FIX. Coadministration of mycophenolate mofetil and tacrolimus with vector does not induce toxicity and does not impair AAV transduction or FIX synthesis. These findings enable a clinical study to assess the effects of immunomodulation on long-term FIX expression in patients with hemophilia B.

Hemophilia A, a deficiency of functional coagulation factor VIII (FVIII), is treated via protein replacement therapy. Restoring 1% to 5% of normal blood FVIII activity prevents spontaneous bleeding, making the disease an attractive gene therapy target. Previously, we have demonstrated short-term activity of a liver-specific AAV2 vector expressing canine B-domain-deleted FVIII (cFVIII) in a hemophilia canine model. Here, we report the long-term efficacy and safety of AAV-cFVIII vectors of serotypes 2, 5, 6, and 8 in both hemophilia A mice and dogs. AAV6-cFVIII and AAV8-cFVIII restored physiologic levels of plasma FVIII activity in hemophilia A mice. The improved efficacy is attributed to more efficient gene transfer in liver compared with AAV2 and AAV5. However, supraphysiologic cFVIII levels correlated with the formation of cFVIII-neutralizing antibodies in these mice. Of importance, hemophilia A dogs that received AAV2-cFVIII, AAV6-cFVIII, and AAV8-cFVIII have persistently expressed therapeutic levels of FVIII, without antibody formation or other toxicities, for more than 3 years. However, liver transduction efficiencies are similar between AAV2, AAV6, and AAV8 serotypes in hemophilia A dogs, in contrast to mice. In summary, this is the first report demonstrating multiyear therapeutic efficacy and safety of multiple AAV-cFVIII vectors in hemophilia A dogs and provides the basis for human clinical studies.

Long-term cures of hemophilia B have been achieved using AAV2 delivering the factor IX gene to the liver of adeno-associated virus (AAV)-naive hemophilic animals. However, the clinical success of this approach requires overcoming pre-existing AAV neutralizing antibodies prevalent in humans. To better define the inhibition of neutralizing antibodies on AAV2-mediated liver transduction, we developed an in vivo passive immunity model. SCID mice were first reconstituted to a defined neutralizing titer with pooled plasma-derived human immunoglobulin. AAV2-FIX vectors then were administered to the liver, and the transduction efficiency was measured by plasma FIX levels. Unexpectedly, AAV2 neutralizing titers lower than 1:10 were sufficient to neutralize 4 to 20 x 10(12) vg/kg of AAV2 vectors in vivo, a capacity that was underestimated by in vitro neutralizing assays. We also evaluated strategies to evade neutralization, including the use of alternative delivery routes, infusion parameters, empty capsids, and alternative AAV serotypes 6 and 8. The results indicate that low AAV2 neutralizing titers can be inhibitory to the tested human and primate AAV vectors delivered into the circulatory system. Therefore, novel nonprimate AAV vectors or compartmentalized delivery may offer more consistent therapeutic effects in the presence of pre-existing AAV neutralizing antibodies.

Additional therapeutic options are needed for patients with bleeding disorders such as hemophilia A, hemophilia B, severe von Willebrand disease, and other rare factor deficiencies. A novel approach to improve coagulation in such clotting disorders has been identified that, parodoxically, involves heparinlike sulfated polysaccharides. Select molecules of this broad class are largely devoid of anticoagulant activity and are here denoted Non-Anticoagulant Sulfated Polysaccharides (NASPs). A mechanism involving blockade of the extrinsic pathway downregulator, Tissue Factor Pathway Inhibitor (TFPI) by NASPs, was conceived as an approach for improving procoagulant behavior in hemophilic settings. A subset of NASPs, including pentosan polysulfate (PPS) and fucoidan inhibited both full-length and Kunitz 1 and 2 (K1K2) TFPI and, at concentrations from 4-500 nM, improved (i.e. accelerated) the clotting time of human hemophilia A and hemophilia B plasmas or plasma with reduced factor VII levels when tested in dilute prothrombin time (dPT) assays. Fucoidan did not reduce normal plasma APTT times implying specificity for extrinsic pathway control. Improved hemostasis in vivo was observed in mice with hemophilias A or B following low dose subcutaneous administration of PPS or fucoidan, or a combination of NASP plus factor supplement. Increased survival of factor deficient mice following a bleeding challenge was observed. Accordingly, administration of select NASP(s), via mechanism(s) not fully understood, represents a unique means of improving coagulation in bleeding disorders.

Using separate adeno-associated viral 2 (AAV2) vectors to deliver the heavy and light chains of factor VIII (FVIII) we have overcome the packaging limitations of AAV, achieving phenotypic correction of hemophilia A in mice. AAV vectors were constructed that use a liver-specific promoter and the cDNA sequences of either the human or canine heavy and light chains of FVIII. After intraportal vein injection of these vectors in hemophilia-A mice, therapeutic to superphysiologic levels of active FVIII were achieved in plasma in a dose-dependent manner. Phenotypic correction of the bleeding diathesis was demonstrated by survival of all treated mice after tail clipping. Biochemical analysis demonstrated lower levels of heavy-chain (25- to 100-fold) compared with light-chain protein in the plasma of treated animals. Differences in gene transfer and transcription did not account for the differences in protein expression. We hypothesize that improvements in FVIII activity could be achieved by improvements in FVIII heavy-chain expression. This work demonstrates that cotransduction of liver with AAV vectors expressing the heavy and light chains of FVIII corrects hemophilia A in vivo, providing an alternative approach to the use of a single vector. This strategy may potentially be useful for other large therapeutic proteins that contain functionally distinct domains.

Gene therapy for hemophilia A requires efficient delivery of the factor VIII gene and sustained protein expression at circulating levels of at least 1% to 2% of normal. Adeno-associated viral type 2 (AAV2) vectors have a number of advantages over other viral vectors, including an excellent safety profile and persistent gene expression. However, a major disadvantage is their small packaging capacity, which has hampered their use in treating diseases such as hemophilia A, cystic fibrosis, and muscular dystrophy, which are caused by mutations in large genes. Here we demonstrate that this can be overcome by using small regulatory elements to drive expression of a B-domain-deleted form of FVIII. The use of this vector for hepatic gene transfer in a canine model of hemophilia A resulted in the sustained (> 14 months) expression of biologically active FVIII. FVIII activity levels of 2% to 4% were achieved. These levels correlated with a partial correction in the whole-blood clotting time and cuticle bleeding time. In addition, immunoprecipitation analysis demonstrated the expression of canine FVIII of the predicted size in the plasma of injected animals. These data support the use of AAV2 vectors in human clinical trials to treat hemophilia A patients.

The MUC1 mucin, lactadherin, and butyrophilin are 3 major components of the human milk fat globule membrane. The mucin inhibits binding of S-fimbriated Escherichia coli to buccal epithelial cells, and lactadherin prevents symptomatic rotavirus infection in breast-fed infants. Butyrophilin has been suggested to be a structural component of the human milk fat globule (HMFG) membrane and to have receptor functions, but has no known anti-infective activity. These HMFG glycoproteins also are present in skimmed milk, possibly associated with phospholipid micelles, while mucin is also in a soluble form. Mucin and lactadherin resist digestion in the stomach of milk-fed infants, while butyrophilin is rapidly degraded. The MUC1 mucin is an extended rod-like structure forming part of the glycocalyx on the surface of many epithelial cells and membranes of milk, and may act as a decoy for binding of infective agents. The extracellular segment of butyrophilin has homology to Ig superfamily receptors and an intracellular domain with homology to developmentally regulated proteins. Lactadherin is a laterally mobile cell adhesion molecule that interacts with integrins and has a novel means of membrane-association involving specific binding to phosphatidylserine. The structural and functional aspects of these glycoproteins are discussed with regard to their role in human milk for breast-fed infants.

Human milk contains many components that protect the newborn against infection at a time when the infant's own defense mechanisms are poorly developed. Fat is one of the major nutrients in human milk. The fat is contained within milk fat globules composed of a core of triglyceride and a membrane consisting of phospholipids, cholesterol, proteins, and glycoproteins. Both the membrane and the core components can provide protection against microorganisms. The major protective membrane glycoproteins, mucin, and lactadherin are resistant to conditions in the newborn's stomach and maintain their structure and function even at low pH and in the presence of the proteolytic enzyme pepsin. The core triglycerides upon hydrolysis by digestive lipases (especially gastric lipase, which is well developed in the newborn) produce free fatty acids and monoglycerides, amphiphylic substances able to lyse enveloped viruses, bacteria, and protozoa. Therefore, in addition to its nutritional value, the fat in human milk has a major protective function.

Human milk fat globule (HMFG) glycoproteins can prevent infections by microorganisms in breast-fed infants; the MUC-1 mucin inhibits binding of S-fimbriated Escherichia coli to buccal mucosa, and lactadherin may prevent symptomatic rotavirus infections. In this study, the survival of these HMFG glycoproteins in the stomach of human milk-fed preterm infants (gestational age = 27.5 +/- 0.4 wk) was assessed, and levels in their mothers' milk determined, using specific RIAs. Butyrophilin, a major component of HMFG membrane that has no demonstrated antimicrobial activity, was studied for comparison. The levels of mucin, lactadherin, and butyrophilin in 41 milk samples of 20 mothers were 729 +/- 75, 93 +/- 10, and 41 +/- 3 microg/mL, respectively. Mucin and lactadherin were significantly higher in early milk samples (<15 d postpartum) than in later milk samples (15-90 d postpartum), whereas butyrophilin showed no such difference. Significant amounts of mucin and lactadherin were found in almost all gastric aspirates of human milk-fed infants, even 4 h after feeding (mucin, 270 +/- 30 microg/mL; lactadherin, 23.2 +/- 4.4 microg/mL), whereas butyrophilin was rapidly degraded in the majority of aspirates. Western blot analysis demonstrated that the immunoreactive mucin, lactadherin, and butyrophilin in the milk-fed gastric aspirates had the expected native molecular weights. Mucin and lactadherin survived at all gastric pH values, whereas butyrophilin was found only at pH > 4. Neither lactadherin nor butyrophilin were detected in gastric aspirates of formula-fed infants (gestational age = 27.8 +/- 0.5 wk), whereas the very low level of mucin (9.1 +/- 1.1 microg/mL) in this group is presumably cross-reacting gastric mucin. These results demonstrate that two HMFG glycoproteins implicated in prevention of infection, MUC-1 mucin and lactadherin, survive and maintain their integrity in the stomachs of human milk-fed preterm infants.

Background: Human milk contains a 46 kDa mucin-associated glycoprotein, lactadherin, which binds specifically to rotavirus and inhibits its replication. This study tested the hypothesis that lactadherin protects against symptoms of rotavirus infection., Methods: 200 infants in Mexico City were recruited at birth and monitored by regular stool EIA for rotavirus, serology, and recording of feeding and stool patterns. Milk samples were obtained from the mothers weekly until 4 weeks post partum then monthly. The sample taken immediately before an infant's episode of rotavirus infection was assayed for lactadherin, butyrophilin, mucin, and secretory IgA. An infection was defined as symptomatic if diarrhoea occurred in the 5 days before or after detection of the virus., Findings: 31 infants developed rotavirus infection; 15 were symptomatic and 16 had no symptoms. The median concentration of lactadherin in the milk samples (obtained 4-41 days [median 13] before the infection) was 48.4 (range 5.6-180) microg/mL in the asymptomatic group and 29-2 (6.2-103-4) microg/mL in the symptomatic group. Although these medians did not differ significantly, in logistic regression analysis adjusted for age at infection and secretory IgA concentration there was a significant difference between the groups (p=0O01). No association between symptom status and concentrations of butyrophilin, mucin, or secretory IgA was found., Interpretation: Protection against rotavirus by human milk is associated with the glycoprotein lactadherin. This association is independent of products of the secretory immune system.

Lactadherin, a major glycoprotein of the human milk fat globule membrane, is abundant in human breast milk and expressed in human breast carcinomas. Previously, we have shown that the mature protein, formerly known as BA46, has three domains: an epidermal growth factor (EGF)-like domain containing an Arg-Gly-Asp (RGD) cell adhesion sequence and C1 and C2 domains similar to those found in coagulation factors V and VIII. An alignment of lactadherin with its bovine (MGP57/53) and murine (MFG-E8) homologs shows that the RGD sequence has been conserved during evolution, suggesting that the RGD sequence is not fortuitous. We demonstrate that lactadherin purified using Triton X-114 phase partitioning promotes RGD-dependent cell attachment of green monkey kidney cells (MA104), mouse fibroblast cells (3T3-L1), and breast carcinoma cells (ELL-G). A lactadherin-specific monoclonal antibody, Mc3, inhibits attachment to purified lactadherin, suggesting that contaminants in the purification are not responsible for binding. In addition, the anti-integrin alpha(v)beta3 monoclonal antibody LM609 inhibits cell attachment of MA104 cells to lactadherin. These results demonstrate that lactadherin promotes RGD-dependent cell adhesion via integrins. Denaturation of lactadherin with heat and reducing conditions diminished cell attachment, suggesting that optimal cell attachment to RGD is dependent on the structural presentation of the sequence.