Live attenuated Salmonella enterica serovar Choleraesuis vector delivering a virus-like particles induces a protective immune response against porcine circovirus type 2 in mice

The virus-like particles (VLPs) of porcine circovirus type 2 (PCV2) is an attractive vaccine candidate that retains the natural conformation of the virion but lacks the viral genome to replicate, thus balancing safety and immunogenicity. However, the assembly of VLPs requires cumbersome subsequent processes, hindering the development of related vaccines. In addition, as a subunit antigen, VLPs are defective in inducing cellular and mucosal immune responses. In this study, the capsid (Cap) protein of PCV2 was synthesized and self-assembled into VLPs in the recombinant attenuated S. Choleraesuis vector, rSC0016(pS-Cap). Furthermore, rSC0016(pS-Cap) induced a Cap-specific Th1-dominant immune response, mucosal immune responses, and neutralizing antibodies against PCV2. Finally, the virus genome copies in mice immunized with the rSC0016(pS-Cap) were significantly lower than those of the empty vector control group after challenge with PCV2. In conclusion, our study demonstrates the potential of using S. Choleraesuis vectors to delivery VLPs, providing new ideas for the development of PCV2 vaccines.     

Transcutaneous immunization of recombinant Staphylococcal enterotoxin B protein using a dissolving microneedle provides potent protection against lethal enterotoxin challenge

Staphylococcal enterotoxin B (SEB) produced by the Staphylococcus aureus bacteriumis most commonly associated with food poisoning and is known to also cause toxic shock syndrome. Currently, no approved vaccine or specific drug is available to treat SEB intoxication. In this study, we fabricated dissolving microneedles (MNs) loaded with recombinant SEB (rSEB) protein, and evaluated its characteristics, including dissolution profile, protein particle size, insertion depth, antigen retention time in vivo, and skin irritation. Our results showed that rSEB protein-loaded dissolving MNs made of chondroitin sulfate (2%) and trehalose (0.8%) could easily penetrate into the mouse skin within 5 min. The rSEB particle size was unchanged before and after MN fabrication. The skin penetration depth of the MNs was 260 µm. Moreover, the MNs also significantly extended the antigen retention time in vivo. rSEB protein-loaded dissolving MNs also triggered slight erythema at the beginning of administration, but this erythema disappeared within a few hours. More importantly, we investigated the immunogenicity and protective efficacy of rSEB protein-loaded dissolving MNs. Challenge studies in mice revealed that mice in full-dose MN group had a high level of SEB specific antibody response thatprovided100% protection against a lethal SEB toxin challenge. However, there was only 60% protection observed in mice that were in the half-dose MN (dose sparing) group. We also determined the pathological alterations in the tissues of the immunized mice. Taken together, these dissolving MNs may present a promising transcutaneous immunization strategy for treating SEB intoxication.     

Transcutaneous immunization via dissolving microneedles protects mice from lethal influenza H7N9 virus challenge

Avian influenza H7N9 virus has first emerged in 2013 and since then has spread in China in five seasonal waves. In humans, influenza H7N9 virus infection is associated with a high fatality rate; thus, an effective vaccine for this virus is needed. In the present study, we evaluated the usefulness of dissolving microneedles (MNs) loaded with influenza H7N9 vaccine in terms of the dissolution time, insertion capacity, insertion depth, and structural integrity of H7N9 virus in vitro. Our in vitro results showed MNs dissolved within 6 mins. The depth of skin penetration was 270 µm. After coating with a matrix material solution, the H7N9 proteins were agglomerated. We detected the H7N9 delivery time and humoral immune response in vivo. In a mouse model, the antigen retention time was longer for MNs than for intramuscular (IM) injection. The humoral response showed that similar to IM administration, MN administration increased the levels of functional and systematic antibodies and protection against the live influenza A/Anhui/01/2013 virus (Ah01/H7N9). The protection level was determined by the analysis of pathological sections of infected lungs. MN and IM administration yielded results superior to those in the control group. Taken together, these findings demonstrate that the use of dissolving MNs to deliver influenza H7N9 vaccines is a promising immunization approach.     

RelCoVax®, a two antigen subunit protein vaccine candidate against SARS-CoV-2 induces strong immune responses in mice

The COVID-19 pandemic has spurred an unprecedented movement to develop safe and effective vaccines against the SARS-CoV-2 virus to immunize the global population. The first set of vaccine candidates that received emergency use authorization targeted the spike (S) glycoprotein of the SARS-CoV-2 virus that enables virus entry into cells via the receptor binding domain (RBD). Recently, multiple variants of SARS-CoV-2 have emerged with mutations in S protein and the ability to evade neutralizing antibodies in vaccinated individuals. We have developed a dual RBD and nucleocapsid (N) subunit protein vaccine candidate named RelCoVax® through heterologous expression in mammalian cells (RBD) and E. coli (N). The RelCoVax® formulation containing a combination of aluminum hydroxide (alum) and a synthetic CpG oligonucleotide as adjuvants elicited high antibody titers against RBD and N proteins in mice after a prime and boost dose regimen administered 2 weeks apart. The vaccine also stimulated cellular immune responses with a potential Th1 bias as evidenced by increased IFN-γ release by splenocytes from immunized mice upon antigen exposure particularly N protein. Finally, the serum of mice immunized with RelCoVax® demonstrated the ability to neutralize two different SARS-CoV-2 viral strains in vitro including the Delta strain that has become dominant in many regions of the world and can evade vaccine induced neutralizing antibodies. These results warrant further evaluation of RelCoVax® through advanced studies and contribute towards enhancing our understanding of multicomponent subunit vaccine candidates against SARS-CoV-2.     

Covax-19/Spikogen® vaccine based on recombinant spike protein extracellular domain with Advax-CpG55.2 adjuvant provides single dose protection against SARS-CoV-2 infection in hamsters

COVID-19 presents an ongoing global health crisis. Protein-based COVID-19 vaccines that are well-tolerated, safe, highly-protective and convenient to manufacture remain of major interest. We therefore sought to compare the immunogenicity and protective efficacy of a number of recombinant SARS-CoV-2 spike protein candidates expressed in insect cells. By comparison to a full length (FL) spike protein detergent-extracted nanoparticle antigen, the soluble secreted spike protein extracellular domain (ECD) generated higher protein yields per liter of culture and when formulated with either Alum-CpG55.2 or Advax-CpG55.2 combination adjuvants elicited robust antigen-specific humoral and cellular immunity in mice. In hamsters, the spike ECD when formulated with either adjuvant induced high serum neutralizing antibody titers even after a single dose. When challenged with the homologous SARS-CoV-2 virus, hamsters immunized with the adjuvanted spike ECD exhibited reduced viral load in day 1–3 oropharyngeal swabs and day 3 nasal turbinate tissue and had no recoverable infectious virus in day 3 lung tissue. The reduction in lung viral load correlated with less weight loss and lower lung pathology scores. The formulations of spike ECD with Alum-CpG55.2 or Advax-CpG55.2 were protective even after just a single dose, although the 2-dose regimen performed better overall and required only half the total amount of antigen. Pre-challenge serum neutralizing antibody levels showed a strong correlation with lung protection, with a weaker correlation seen with nasal or oropharyngeal protection. This suggests that serum neutralizing antibody levels may correlate more closely with systemic, rather than mucosal, protection. The spike protein ECD with Advax-CpG55.2 formulation (Covax-19® vaccine) was selected for human clinical development.     

Immunogenicity and protective efficacy of adenoviral and subunit RSV vaccines based on stabilized prefusion F protein in pre-clinical models

Respiratory Syncytial Virus (RSV) remains a leading cause of severe respiratory disease for which no licensed vaccine is available. We have previously described the derivation of an RSV Fusion protein (F) stabilized in its prefusion conformation (preF) as vaccine immunogen and demonstrated superior immunogenicity in naive mice of preF versus wild type RSV F protein, both as protein and when expressed from an Ad26 vaccine vector. Here we address the question if there are qualitative differences between the two vaccine platforms for induction of protective immunity. In naïve mice, both Ad26.RSV.preF and preF protein induced humoral responses, whereas cellular responses were only elicited by Ad26.RSV.preF. In RSV pre-exposed mice, a single dose of either vaccine induced cellular responses and strong humoral responses. Ad26-induced RSV-specific cellular immune responses were detected systemically and locally in the lungs. Both vaccines showed protective efficacy in the cotton rat model, but Ad26.RSV.preF conferred protection at lower virus neutralizing titers in comparison to RSV preF protein. Factors that may contribute to the protective capacity of Ad26.RSV.preF elicited immunity are the induced IgG2a antibodies that are able to engage Fcγ receptors mediating Antibody Dependent Cellular Cytotoxicity (ADCC), and the induction of systemic and lung resident RSV specific CD8 + T cells. These data demonstrate qualitative improvement of immune responses elicited by an adenoviral vector based vaccine encoding the RSV preF antigen compared to the subunit vaccine in small animal models which may inform RSV vaccine development.     

Phosphorylcholine intranasal immunization with a 13-valent pneumococcal conjugate vaccine can boost immune response against Streptococcus pneumoniae

ObjectiveThis study aimed to investigate whether systemic immunization with a 13-valent pneumococcal conjugate vaccine (PCV13) followed by intranasal (IN) immunization with phosphorylcholine (PC) can boost immune response against Streptococcus pneumoniae.Materials and methodsTwo weeks after the intraperitoneal (IP) injection of PCV13, mice were divided into two groups (mice requiring another IP injection of PCV13 and mice requiring PC-keyhole limpet hemocyanin IN immunization in combination with cholera toxin as a mucosal adjuvant) to compare the magnitude of systemic and mucosal immune responses against S. pneumoniae and PC.ResultsSerum immunoglobulin (Ig) G antibody titer against the vaccine strains of S. pneumoniae was similar between the PCV13 systemic immunization group and PC IN immunization group, while the serum IgG antibody titer against PC was significantly higher in the PC IN immunization group. PC-specific IgA antibody titer in the nasal lavage and PC-specific IgA-producing cell number in the nasal mucosa were also significantly higher in the PC IN immunization group. Induction of PC-specific IgA in the PC IN immunization group enhanced the clearance of bacteria from the middle ear.ConclusionAdditional IN immunization with PC after PCV13 immunization, which is currently conducted under a periodic vaccination program, can produce a booster effect comparable to that achieved by additional systemic immunization as well as PC-specific mucosal immune response, thereby providing protection against S. pneumoniae serotypes not contained in PCV13.     

Protection of vaccination versus hybrid immunity against infection with COVID-19 Omicron variants among Health-Care Workers

Background aimThe Omicron COVID-19 variants BA.1* and BA.2* evade immune system leading to increased transmissibility and breakthrough infections. We aim to test the hypothesis that immunity achieved post COVID-19 infection combined with vaccination (hybrid immunity), is more effective against Omicron infection than vaccination alone in a health-care setting.MethodsData on regular pre-emptive PCR testing from all Health-Care Workers (HCWs) at Laiko University Hospital from 29th December 2020, date on which the national COVID-19 immunization program began in Greece, until 24th May 2022, were retrospectively collected and recorded. The infection rate was calculated after December 21st, 2021, when Omicron was the predominant circulating variant in Greece, as the total number of infections (positive PCR COVID-19 test regardless of symptoms) divided by the total person-months at risk.ResultsOf 1,305 vaccinated HCWs who were included in the analysis [median age of 47 (IQR: 36, 56) years, 66.7 % women], 13 % and 87 % had received 2 or 3 vaccine doses (full and booster vaccination), respectively. A COVID-19 infection had occurred in 135 of 1,305 of participants prior to Omicron predominance. Of those 135 HCWs with hybrid immunity only 13 (9.6 %) were re-infected. Of the 154 and 1,016 HCWs with full and booster vaccination-induced immunity, respectively, 71 (46.1 %, infection rate 13.4/100 person-months) and 448 (44.1 %, infection rate 12.2/100 person-months) were infected during the follow up period. No association between gender or age and COVID-19 infection was found and none of the participants had a severe infection or died.ConclusionsHybrid immunity confers higher protection by almost 5-fold compared to full or booster vaccination for COVID-19 infection with the Omicron variant among HCWs who are at high risk of exposure. This may inform public health policies on how to achieve optimal immunity in terms of the timing and mode of vaccination.     

The added effect of non-pharmaceutical interventions and lifestyle behaviors on vaccine effectiveness against severe COVID-19 in Chile: A matched case-double control study

BackgroundWorld Health Organization approved vaccines have demonstrated relatively high protection against moderate to severe COVID-19. Prospective vaccine effectiveness (VE) designs with first-hand data and population-based controls are nevertheless rare. Neighborhood compared to hospitalized controls, may differ in compliance to non-pharmacuetical interventions (NPI) compliance, which may influence VE results in real-world settings. We aimed to determine VE against COVID-19 intensive-care-unit (ICU) admission using hospital and community-matched controls in a prospective design.MethodsWe conducted a multicenter, observational study of matched cases and controls (1:3) in adults ≧18 years of age from May to July 2021. For each case, a hospital control and two community controls were matched by age, gender, and hospital admission date or neighborhood of residence. Conditional logistic regression models were built, including interaction terms between NPIs, lifestyle behaviors, and vaccination status; the model’s β coefficients represent the added effect these terms had on COVID-19 VE.ResultsCases and controls differed in several factors including education level, obesity prevalence, and behaviors such as compliance with routine vaccinations, use of facemasks, and routine handwashing. VE was 98·2% for full primary vaccination and 85·6% for partial vaccination when compared to community controls, and somewhat lower, albeit not significantly, compared to hospital controls. A significant added effect to vaccination in reducing COVID-19 ICU admission was regular facemask use and VE was higher among individuals non-compliant with the national vaccine program, and/or tonroutine medical visits during the prior year.ConclusionVE against COVID-19 ICU admission in this stringent prospective case-double control study reached 98% two weeks after full primary vaccination, confirming the high effectiveness provided by earlier studies. Face mask use and hand washing were independent protective factors, the former adding additional benefit to VE. VE was significantly higher in subjects with increased risk behaviors.     

A novel combined vaccine against classical swine fever and porcine epidemic diarrhea viruses elicits a significant Th2-favored humoral response in mice

Many Chinese breeding pigs are repeatedly vaccinated against classical swine fever virus (CSFV) and porcine epidemic diarrhea virus (PEDV), which cause fatal, highly contagious diseases. To reduce their high frequency vaccination-induced immune stress, we constructed a combined vaccine based on the E2 protein of CSFV and the S1 spike protein subunit of PEDV (named E2-S1). In mice, the E2-S1 vaccine elicited higher neutralizing antibody titers and IgG1/IgG2a ratios against CSFV and PEDV than those induced by individual E2 or S1 vaccines. Moreover, it elicited high IL-4 expression, but no IFN-γ expression. The results suggest that good compatibility exists between E2 and S1 antigens, and the E2-S1 vaccine can elicit a strong Th2-type cell-mediated humoral immune response. The E2-S1 recombinant fusion protein provides a novel vaccine candidate against both CSFV and PEDV, laying the foundation for future combination vaccines against swine diseases.     

Glucopyranosyl lipid adjuvant enhances immune response to Ebola virus-like particle vaccine in mice

The identification of adjuvants that promote lasting antigen-specific immunity and augment vaccine efficacy are integral to the development of new protein-based vaccines. The Ebola virus-like particle (VLP) vaccine expressing Ebola virus glycoprotein (GP) and matrix protein (VP40) was used in this study to evaluate the ability of TLR4 agonist glucopyranosyl lipid adjuvant (GLA) formulated in a stable emulsion (SE) to enhance immunogenicity and promote durable protection against mouse-adapted Ebola virus (ma-EBOV). Antibody responses and Ebola-specific T cell responses were evaluated post vaccination. Survival analysis after lethal ma-EBOV challenge was performed 4 weeks and 22 weeks following final vaccination. GLA-SE enhanced EBOV-specific immunity and resulted in long-term protection against challenge with ma-EBOV infection in a mouse model. Specifically, GLA-SE elicited Th1-skewed antibodies and promoted the generation of EBOV GP-specific polyfunctional T cells. These results provide further support for the utility of TLR4 activating GLA-SE-adjuvanted vaccines.     

Endpoint and epitope-specific antibody responses as correlates of vaccine-mediated protection of mice against ricin toxin

The successful licensure of vaccines for biodefense is contingent upon the availability of well-established correlates of protection (CoP) in at least two animal species that can be applied to humans, without the need to assess efficacy in the clinic. In this report we describe a multivariate model that combines pre-challenge serum antibody endpoint titers (EPT) and values derived from an epitope profiling immune-competition capture (EPICC) assay as a predictor in mice of vaccine-mediated immunity against ricin toxin (RT), a Category B biothreat. EPICC is a modified competition ELISA in which serum samples from vaccinated mice were assessed for their ability to inhibit the capture of soluble, biotinylated (b)-RT by a panel of immobilized monoclonal antibodies (mAbs) directed against four immunodominant toxin-neutralizing regions on the enzymatic A chain (RTA) of RT. In a test cohort of mice (n = 40) vaccinated with suboptimal doses of the RTA subunit vaccine, RiVax®, we identified two mAbs, PB10 and SyH7, which had EPICC inhibition values in pre-challenge serum samples that correlated with survival following a challenge with 5 × LD₅₀ of RT administered by intraperitoneal (IP) injection. Analysis of a larger cohort of mice (n = 645) revealed that a multivariate model combining endpoint titers and EPICC values for PB10 and SyH7 as predictive variables had significantly higher statistical power than any one of the independent variables alone. Establishing the correlates of vaccine-mediated protection in mice represents an important steppingstone in the development of RiVax® as a medical countermeasure under the United States Food and Drug Administration’s “Animal Rule.”     

Vaccination against cocaine using a modifiable dendrimer nanoparticle platform

Pharmacological therapies for the treatment of cocaine addiction have had disappointing efficacy, and the lack of recent developments in the clinical care of cocaine-addicted patients indicates a need for novel treatment strategies. Recent studies have shown that vaccination against cocaine to elicit production of antibodies that reduce concentrations of free drug in the blood is a promising method to protect against the effects of cocaine and reduce rates of relapse. However, the poorly immunogenic nature of cocaine remains a major hurdle to active immunization. Therefore, we hypothesized that strategies to increase targeted exposure of cocaine to the immune system may produce a more effective vaccine. To specifically direct an immune response against cocaine, in the present study we have conjugated a cocaine analog to a dendrimer-based nanoparticle carrier with MHC II-binding moieties that previously has been shown to activate antigen-presenting cells necessary for antibody production. This strategy produced a rapid, prolonged, and high affinity anti-cocaine antibody response without the need for an adjuvant. Surprisingly, additional evaluation using multiple adjuvant formulations in two strains of inbred mice found adjuvants were either functionally redundant or deleterious in the vaccination against cocaine using this platform. The use of conditioned place preference in rats after administration of this vaccine provided proof of concept for the ability of this vaccine to diminish cocaine reward. Together these data demonstrate the intrinsic efficacy of an immune-targeting dendrimer-based cocaine vaccine, with a vast potential for design of future vaccines against other poorly immunogenic antigens by substitution of the conjugated cargo.     

DS-5670a,a novel mRNA-encapsulated lipid nanoparticle vaccine against severe acute respiratory syndrome coronavirus 2: Results from a phase 2 clinical study

BackgroundDS-5670a is a vaccine candidate for coronavirus disease 2019 (COVID-19) harnessing a novel modality composed of messenger ribonucleic acid (mRNA) encoding the receptor-binding domain (RBD) from the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encapsulated in lipid nanoparticles. Here, we report the safety, immunogenicity, and pharmacokinetic profile of DS-5670a from a phase 2 clinical trial in healthy adults who were immunologically naïve to SARS-CoV-2.MethodsThe study consisted of an open-label, uncontrolled, dose-escalation part and a double-blind, randomized, uncontrolled, 2-arm, parallel-group part. A total of 80 Japanese participants were assigned to receive intramuscular DS-5670a, containing either 30 or 60 µg of mRNA, as two injections administered 4 weeks apart. Safety was assessed by characterization of treatment-emergent adverse events (TEAEs). Immunogenicity was assessed by neutralization titers against SARS-CoV-2, anti-RBD immunoglobulin (Ig)G levels, and SARS-CoV-2 spike-specific T cell responses. Plasma pharmacokinetic parameters of DS-5670a were also evaluated.ResultsMost solicited TEAEs were mild or moderate with both the 30 and 60 µg mRNA doses. Four participants (10 %) in the 60 µg mRNA group developed severe redness at the injection site, but all cases resolved without treatment. There were no serious TEAEs and no TEAEs leading to discontinuation. Humoral immune responses in both dose groups were greater than those observed in human convalescent serum; the 60 µg mRNA dose produced better responses. Neutralization titers were found to be correlated with anti-RBD IgG levels (specifically IgG1). DS-5670a elicited antigen-specific T helper 1-polarized cellular immune responses.ConclusionsThe novel mRNA-based vaccine candidate DS-5670a provided favorable immune responses against SARS-CoV-2 with a clinically acceptable safety profile. Confirmatory trials are currently ongoing to evaluate the safety and immunogenicity of DS-5670a as the primary vaccine and to assess the immunogenicity when administered as a heterologous or homologous booster.Trial registry: https://jrct.niph.go.jp/latest-detail/jRCT2071210086.     

The v-safe after vaccination health checker: Active vaccine safety monitoring during CDC’s COVID-19 pandemic response

The Centers for Disease Control and Prevention (CDC) developed and implemented the v-safe after vaccination health checker (v-safe) to monitor COVID-19 vaccine safety and as an active surveillance supplement to existing CDC vaccine safety monitoring programs. V-safe allows persons who received COVID-19 vaccines to report on post-vaccination experiences and how symptoms affected their health at daily, weekly, and monthly timepoints after vaccination. Text message reminders are sent linking to Internet-based health check-in surveys. Surveys include questions to identify v-safe participants who may be eligible to enroll in a separate pregnancy registry activity that evaluates maternal and infant outcomes in those pregnant at the time of vaccination or receiving vaccine in the periconception period.We describe the development of and enhancements to v-safe, data management, promotion and communication to vaccination sites and partners, publications, strengths and limitations, and implications for future systems. We also describe enrollment in v-safe over time and demographics of persons participating in v-safe during the first year of operation (December 14, 2020 – December 13, 2021). During this time, 9,342,582 persons submitted 131,543,087 v-safe surveys. The majority of participants were female (62.3 %) and non-Hispanic White (61.2 %); median age was 49.0 years. Most participants reported receiving an mRNA COVID-19 vaccine as their first recorded dose (95.0 %).V-safe contributed to CDC’s vaccine safety assessments for FDA-authorized COVID-19 vaccines by enabling near real-time reporting of reactogenicity once the COVID-19 vaccination program began in the community, encouraging reports to the Vaccine Adverse Event Reporting System and facilitating enrollment in a large post-vaccination pregnancy registry. Given that v-safe is an integral component of the most comprehensive safety monitoring program in U.S. history, we believe that this approach has promise as a potential application for future pandemic response activities as well as rollout of novel vaccines in a non-pandemic context.     

Willingness to vaccinate children against COVID-19 declined during the pandemic

ObjectivesTo document the level of vaccine hesitancy in caregivers’ of children younger than 12 years of age over the course of the pandemic in Pediatric Emergency Departments (ED).Study designOngoing multicenter, cross-sectional survey of caregivers presenting to 19 pediatric EDs in the USA, Canada, Israel, and Switzerland during first months of the pandemic (phase1), when vaccines were approved for adults (phase2) and most recently when vaccines were approved for children (phase3).ResultsWillingness to vaccinate rate declined over the study period (59.7%, 56.1% and 52.1% in the three phases). Caregivers who are fully vaccinated, who have higher education, and those worried their child had COVID-19 upon arrival to the ED, were more likely to plan to vaccinate in all three phases. Mothers were less likely to vaccinate early in the pandemic, but this hesitancy attenuated in later phases. Older caregivers were more willing to vaccinate, and caregivers of older children were less likely to vaccinate their children in phase 3. During the last phase, willingness to vaccinate was lowest in those who had a primary care provider but did not rely on their advice for medical decisions (34%). Those with no primary care provider and those who do and rely on their medical advice, had similar rates of willingness to vaccinate (55.1% and 52.1%, respectively).ConclusionsCOVID-19 vaccine hesitancy is widespread and growing over time, and public health measures should further try to leverage identified factors associated with hesitancy in order to enhance vaccination rates among children.     

Breadth of humoral immune responses to the C-terminus of the circumsporozoite protein is associated with protective efficacy induced by the RTS,S malaria vaccine

The circumsporozoite protein (CSP) is the main surface antigen of malaria sporozoites, a prime vaccine target, and is known to have polymorphisms in the C-terminal region. Vaccines using a single allele may have lower efficacy against genotypic variants. Recent studies have found evidence suggesting the efficacy of the CSP-based RTS,S malaria vaccine may be limited against P. falciparum CSP alleles that diverge from the 3D7 vaccine allele, particularly in this polymorphic C-terminal region. In order to assess the breadth of the RTS,S-induced antibody responses against CSP C-terminal antigenic variants, we used a novel multiplex assay to measure reactivity of serum samples from a recent RTS,S study against C-terminal peptides from 3D7 and seven additional CSP alleles that broadly represent the genetic diversity found in circulating P. falciparum field isolates. We found that responses to the variants showed, on average, a ~ 30-fold reduction in reactivity relative to the vaccine-matched 3D7 allele. The extent of this reduction, ranging from 21 to 69-fold, correlated with the number of polymorphisms between the variants and 3D7. We calculated antibody breadth of each sample as the median relative reactivity to the seven CSP variants compared to 3D7. Surprisingly, protection from 3D7 challenge in the RTS,S study was associated with higher C-terminal antibody breadth. These findings suggest CSP C-terminal-specific avidity or fine-specificity may play a role in RTS,S-mediated protection and that breadth of C-terminal CSP-specific antibody responses may be a marker of protection.