ABSTRACT
Live vaccines have emerged as one of the most potentially cost-effective measures for the control of bovine neosporosis. Previous studies have shown that Nc-Spain 1H is a naturally attenuated isolate of Neospora caninum and that immunisation with live Nc-Spain 1H tachyzoites generated a protective immune response in mice. The aim of this study was to evaluate the safety and efficacy of immunisation in cattle. N. caninum-seronegative heifers were immunised subcutaneously twice with 107 live Nc-Spain 1H tachyzoites prior to artificial insemination. No adverse reactions or negative effects on reproductive parameters were recorded following immunisation. In immunised and non-challenged heifers, no foetal deaths were observed, and none of the calves was congenitally infected. The efficacy against N. caninum-associated foetal death and vertical transmission was determined after challenge with high doses of the Nc-1 isolate at 70 and 135 days of gestation, respectively. After the challenge in early gestation, the immunisation induced a protection of 50% against foetal death. In addition, the microsatellite analysis performed in PCR-positive tissue samples from foetuses that died after challenge infection showed that the profiles corresponded to the challenge isolate Nc-1. A degree of protection against vertical transmission was observed after challenge at mid-gestation; calves from immunised heifers showed significantly lower pre-colostral Neospora-specific antibody titres than calves from the non-immunised/challenge group (P < 0.05). Strong antibody and interferon gamma responses were induced in the immunised heifers. This study indicates that the immunisation before pregnancy with the Nc-Spain 1H vaccine isolate appeared to be safe and reduced the occurrence of N. caninum-associated abortion and vertical transmission in experimentally infected cattle. In light of these encouraging results, the next step for testing this live attenuated candidate should be the assessment of its efficacy and safety in naturally infected cattle.
Subject(s)
Cattle Diseases/prevention & control , Coccidiosis/veterinary , Infectious Disease Transmission, Vertical/veterinary , Neospora/immunology , Protozoan Vaccines/immunology , Animals , Antibodies, Protozoan/blood , Cattle , Cattle Diseases/immunology , Coccidiosis/immunology , Coccidiosis/prevention & control , Female , Infectious Disease Transmission, Vertical/prevention & control , Interferon-gamma/genetics , Interferon-gamma/metabolism , Microsatellite Repeats , Neospora/genetics , Neospora/pathogenicity , Polymerase Chain Reaction/veterinary , Pregnancy , Protozoan Vaccines/administration & dosage , Spain , Vaccines, Attenuated/administration & dosage , Vaccines, Attenuated/immunologyABSTRACT
The continuing high global incidence of COVID-19 and the undervaccinated status of billions of persons strongly motivate the development of a new generation of efficacious vaccines. We have developed an adjuvanted vaccine candidate, PHH-1V, based on a protein comprising the receptor binding domain (RBD) of the Beta variant of SARS-CoV-2 fused in tandem with the equivalent domain of the Alpha variant, with its immunogenicity, safety and efficacy previously demonstrated in mouse models. In the present study, we immunized pigs with different doses of PHH-1V in a prime-and-boost scheme showing PHH-1V to exhibit an excellent safety profile in pigs and to produce a solid RBD-specific humoral response with neutralising antibodies to 7 distinct SARS-CoV-2 variants of concern, with the induction of a significant IFNγ+ T-cell response. We conclude that PHH-1V is safe and elicits a robust immune response to SARS-CoV-2 in pigs, a large animal preclinical model.
Subject(s)
COVID-19 , Mice , Animals , Swine , COVID-19/prevention & control , SARS-CoV-2 , COVID-19 Vaccines/adverse effects , Antibodies, Viral , Antibodies, Neutralizing , Immunogenicity, Vaccine , Spike Glycoprotein, Coronavirus/geneticsABSTRACT
SARS-CoV-2 emerged in December 2019 and quickly spread worldwide, continuously striking with an unpredictable evolution. Despite the success in vaccine production and mass vaccination programs, the situation is not still completely controlled, and therefore accessible second-generation vaccines are required to mitigate the pandemic. We previously developed an adjuvanted vaccine candidate coded PHH-1V, based on a heterodimer fusion protein comprising the RBD domain of two SARS-CoV-2 variants. Here, we report data on the efficacy, safety, and immunogenicity of PHH-1V in cynomolgus macaques. PHH-1V prime-boost vaccination induces high levels of RBD-specific IgG binding and neutralizing antibodies against several SARS-CoV-2 variants, as well as a balanced Th1/Th2 cellular immune response. Remarkably, PHH-1V vaccination prevents SARS-CoV-2 replication in the lower respiratory tract and significantly reduces viral load in the upper respiratory tract after an experimental infection. These results highlight the potential use of the PHH-1V vaccine in humans, currently undergoing Phase III clinical trials.
ABSTRACT
In response to COVID-19 pandemic, we have launched a vaccine development program against SARS-CoV-2. Here we report the safety, tolerability, and immunogenicity of a recombinant protein RBD fusion heterodimeric vaccine against SARS-CoV-2 (PHH-1V) evaluated in a phase 1-2a dose-escalation, randomized clinical trial conducted in Catalonia, Spain. 30 young healthy adults were enrolled and received two intramuscular doses, 21 days apart of PHH-1V vaccine formulations [10 µg (n = 5), 20 µg (n = 10), 40 µg (n = 10)] or control [BNT162b2 (n = 5)]. Each PHH-1V group had one safety sentinel and the remaining participants were randomly assigned. The primary endpoint was solicited events within 7 days and unsolicited events within 28 days after each vaccination. Secondary endpoints were humoral and cellular immunogenicity against the variants of concern (VOCs) alpha, beta, delta and gamma. All formulations were safe and well tolerated, with tenderness and pain at the site of injection being the most frequently reported solicited events. Throughout the study, all participants reported having at least one mild to moderate unsolicited event. Two unrelated severe adverse events (AE) were reported and fully resolved. No AE of special interest was reported. Fourteen days after the second vaccine dose, all participants had a >4-fold change in total binding antibodies from baseline. PHH-1V induced robust humoral responses with neutralizing activities against all VOCs assessed (geometric mean fold rise at 35 days p < 0.0001). The specific T-cell response assessed by ELISpot was moderate. This initial evaluation has contributed significantly to the further development of PHH-1V, which is now included in the European vaccine portfolio.ClinicalTrials.gov Identifier NCT05007509EudraCT No. 2021-001411-82.
ABSTRACT
Background: A SARS-CoV-2 protein-based heterodimer vaccine, PHH-1V, has been shown to be safe and well-tolerated in healthy young adults in a first-in-human, Phase I/IIa study dose-escalation trial. Here, we report the interim results of the Phase IIb HH-2, where the immunogenicity and safety of a heterologous booster with PHH-1V is assessed versus a homologous booster with BNT162b2 at 14, 28 and 98 days after vaccine administration. Methods: The HH-2 study is an ongoing multicentre, randomised, active-controlled, double-blind, non-inferiority Phase IIb trial, where participants 18 years or older who had received two doses of BNT162b2 were randomly assigned in a 2:1 ratio to receive a booster dose of vaccine-either heterologous (PHH-1V group) or homologous (BNT162b2 group)-in 10 centres in Spain. Eligible subjects were allocated to treatment stratified by age group (18-64 versus ≥65 years) with approximately 10% of the sample enrolled in the older age group. The primary endpoints were humoral immunogenicity measured by changes in levels of neutralizing antibodies (PBNA) against the ancestral Wuhan-Hu-1 strain after the PHH-1V or the BNT162b2 boost, and the safety and tolerability of PHH-1V as a boost. The secondary endpoints were to compare changes in levels of neutralizing antibodies against different variants of SARS-CoV-2 and the T-cell responses towards the SARS-CoV-2 spike glycoprotein peptides. The exploratory endpoint was to assess the number of subjects with SARS-CoV-2 infections ≥14 days after PHH-1V booster. This study is ongoing and is registered with ClinicalTrials.gov, NCT05142553. Findings: From 15 November 2021, 782 adults were randomly assigned to PHH-1V (n = 522) or BNT162b2 (n = 260) boost vaccine groups. The geometric mean titre (GMT) ratio of neutralizing antibodies on days 14, 28 and 98, shown as BNT162b2 active control versus PHH-1V, was, respectively, 1.68 (p < 0.0001), 1.31 (p = 0.0007) and 0.86 (p = 0.40) for the ancestral Wuhan-Hu-1 strain; 0.62 (p < 0.0001), 0.65 (p < 0.0001) and 0.56 (p = 0.003) for the Beta variant; 1.01 (p = 0.92), 0.88 (p = 0.11) and 0.52 (p = 0.0003) for the Delta variant; and 0.59 (p ≤ 0.0001), 0.66 (p < 0.0001) and 0.57 (p = 0.0028) for the Omicron BA.1 variant. Additionally, PHH-1V as a booster dose induced a significant increase of CD4+ and CD8+ T-cells expressing IFN-γ on day 14. There were 458 participants who experienced at least one adverse event (89.3%) in the PHH-1V and 238 (94.4%) in the BNT162b2 group. The most frequent adverse events were injection site pain (79.7% and 89.3%), fatigue (27.5% and 42.1%) and headache (31.2 and 40.1%) for the PHH-1V and the BNT162b2 groups, respectively. A total of 52 COVID-19 cases occurred from day 14 post-vaccination (10.14%) for the PHH-1V group and 30 (11.90%) for the BNT162b2 group (p = 0.45), and none of the subjects developed severe COVID-19. Interpretation: Our interim results from the Phase IIb HH-2 trial show that PHH-1V as a heterologous booster vaccine, when compared to BNT162b2, although it does not reach a non-inferior neutralizing antibody response against the Wuhan-Hu-1 strain at days 14 and 28 after vaccination, it does so at day 98. PHH-1V as a heterologous booster elicits a superior neutralizing antibody response against the previous circulating Beta and the currently circulating Omicron BA.1 SARS-CoV-2 variants in all time points assessed, and for the Delta variant on day 98 as well. Moreover, the PHH-1V boost also induces a strong and balanced T-cell response. Concerning the safety profile, subjects in the PHH-1V group report significantly fewer adverse events than those in the BNT162b2 group, most of mild intensity, and both vaccine groups present comparable COVID-19 breakthrough cases, none of them severe. Funding: HIPRA SCIENTIFIC, S.L.U.
ABSTRACT
Current COVID-19 vaccines have been associated with a decline in infection rates, prevention of severe disease, and a decrease in mortality rates. However, SARS-CoV-2 variants are continuously evolving, and development of new accessible COVID-19 vaccines is essential to mitigate the pandemic. Here, we present data on preclinical studies in mice of a receptor-binding domain (RBD)-based recombinant protein vaccine (PHH-1V) consisting of an RBD fusion heterodimer comprising the B.1.351 and B.1.1.7 SARS-CoV-2 variants formulated in SQBA adjuvant, an oil-in-water emulsion. A prime-boost immunisation with PHH-1V in BALB/c and K18-hACE2 mice induced a CD4+ and CD8+ T cell response and RBD-binding antibodies with neutralizing activity against several variants, and also showed a good tolerability profile. Significantly, RBD fusion heterodimer vaccination conferred 100% efficacy, preventing mortality in SARS-CoV-2 infected K18-hACE2 mice, but also reducing Beta, Delta and Omicron infection in lower respiratory airways. These findings demonstrate the feasibility of this recombinant vaccine strategy.
ABSTRACT
The parasite Neospora caninum is an important abortifacient agent in cattle worldwide. At present, the development of an effective and safe vaccine against bovine neosporosis is of great relevance. Recently, a new isolate of N. caninum (Nc-Spain 1 H) which was obtained from the brain of an asymptomatic congenitally infected calf, exhibited non-virulent behaviour in mouse and bovine infection models. The aim of this study was to determine the safety and efficacy of Nc-Spain 1 H when used as a vaccinal isolate in well-established BALB/c models of congenital and cerebral neosporosis. Mice were subcutaneously immunised twice at 3-week intervals and were challenged with 2 × 106 tachyzoites of the virulent Nc-Liv isolate. After immunisation with live Nc-Spain 1 H tachyzoites, no parasitic DNA was detected in the dams' brains before challenge and microsatellite analysis performed in PCR-positive mice showed that the profiles corresponded to the challenge isolate Nc-Liv, indicating the Nc-Spain 1 H isolate to be a safe vaccine candidate. The efficacy of the live vaccine was evaluated in the first experiment after the immunisation of mice with 5 × 105 live Nc-Spain 1 H tachyzoites. This immunisation protocol significantly reduced the neonatal mortality to 2.4%, reduced the vertical transmission from 89.1% to 2.3% and completely limited the cerebral infection. These results were associated with a Th1-type immune response. In the second experiment, the effect of various immunising doses was established using ten-fold dilutions of the tachyzoites (from 5 × 105 to 5 × 10). In all the cases, congenital protection rates above 60% were observed, and the mice that were immunised with the lowest dose (5 × 10) presented the highest protection rate (86%). Moreover, low immunising doses of Nc-Spain 1 H induced an IgG2a response, and high parasitic doses induced an IgG1 response. These results evidence the safety and the efficient protection that was conferred by Nc-Spain 1 H against congenital neosporosis, even when the mice were immunised with low parasitic doses.
Subject(s)
Antigens, Protozoan/administration & dosage , Brain/pathology , Coccidiosis/prevention & control , Coccidiosis/veterinary , Neospora/immunology , Protozoan Vaccines/immunology , Animals , Antibodies, Protozoan/blood , Antigens, Protozoan/immunology , Brain/parasitology , Coccidiosis/immunology , Cytokines/genetics , Cytokines/metabolism , DNA, Protozoan/analysis , Dose-Response Relationship, Immunologic , Enzyme-Linked Immunosorbent Assay/veterinary , Female , Immunity, Humoral , Immunoglobulin G/blood , Injections, Subcutaneous/veterinary , Mice , Mice, Inbred BALB C , Neospora/genetics , Neospora/pathogenicity , Polymerase Chain Reaction/veterinary , Pregnancy , Protozoan Vaccines/administration & dosage , Vaccines, Attenuated/administration & dosage , Vaccines, Attenuated/immunologyABSTRACT
Streptococcus uberis is a worldwide pathogen that causes intramammary infections in dairy cattle. Because virulence factors determining the pathogenicity of S. uberis have not been clearly identified so far, a commercial vaccine is not yet available. Different S. uberis strains have the ability to form biofilm in vitro, although the association of this kind of growth with the development of mastitis is unknown. The objective of this study was to evaluate the potential use as vaccine antigens of proteins from S. uberis biofilms, previously identified by proteomic and immunological analyses. The capability of eliciting a protective immune response by targeted candidates was assayed on a murine model. Sera from rabbits immunized with S. uberis biofilm preparations and a convalescent cow intra-mammary infected with S. uberis were probed against cell wall proteins from biofilm and planktonic cells previously separated by two-dimensional gel electrophoresis. Using rabbit immunized serum, two proteins were found to be up-regulated in biofilm cells as compared to planktonic cells; when serum from the convalescent cow was used, up to sixteen biofilm proteins were detected. From these proteins, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), fructose-biphosphate aldolase (FBA), and elongation factor Ts (EFTs) were chosen to be tested as vaccine antigen candidates. For this purpose, different groups of mice were immunized with the three recombinant-expressed proteins (each one formulated separately in a vaccine), and thereafter intraperitoneally challenged with S. uberis. The three proteins induced specific IgG antibodies, but a significant reduction of mortality was only observed in the groups of mice vaccinated with FBA or EFTs. These results suggest that FBA and EFTs might be considered as strong antigenic candidates for a vaccine against S. uberis bovine mastitis. Moreover, this is the first study to indicate that also in S. uberis, GAPDH, FBA and EFTs, as proteins detected in both cytoplasm and cell wall fractions, can play a second function (moonlighting), the latter being particularly involved in the virulence of such a pathogen organism.
Subject(s)
Antigens, Bacterial/immunology , Bacterial Vaccines/immunology , Mastitis, Bovine/prevention & control , Streptococcal Infections/veterinary , Streptococcus , Animals , Antibodies, Bacterial/blood , Biofilms , Cattle , Electrophoresis, Gel, Two-Dimensional , Female , Fructose-Bisphosphatase/immunology , Gene Expression Regulation, Bacterial , Immunoglobulin G/blood , Mice , Mice, Inbred BALB C , Peptide Elongation Factors/immunology , Proteomics , Rabbits , Recombinant Proteins/immunology , Streptococcal Infections/prevention & controlABSTRACT
A genetic screening for osmoregulated genes allowed us to identify the yfeR gene of Salmonella enterica serovar Typhimurium. The yfeR gene product encodes a novel LysR-type transcriptional regulator (LTTR), the expression of which decreases when external osmolarity increases. Out of the adjacent gene yfeH, YfeR modulates expression of several genes that may be required for optimal growth under low osmolarity conditions.
Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/metabolism , DNA, Bacterial/metabolism , Salmonella typhimurium/genetics , Salmonella typhimurium/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Water-Electrolyte Balance/genetics , Binding Sites , DNA, Intergenic/genetics , Gene Expression Regulation, Bacterial , Mutagenesis, Insertional , Protein BindingABSTRACT
In this study, the protection afforded by a Neospora caninum inactivated vaccine formulated with three different adjuvants (water-in-oil emulsion, aluminum hydroxide with CpG oligodeoxynucleotides and aluminum hydroxide with ginseng extract) and three different parasite doses (10(5), 5 × 10(5) or 10(6) inactivated whole tachyzoites) was evaluated using a mouse model. Mice were immunized subcutaneously twice at three-week intervals with inactivated Nc-Spain 1H tachyzoites and challenged by intraperitoneal inoculation with 10(6) live Nc-1 tachyzoites. The efficacy of the immunization was evaluated in non-pregnant BALB/c mice on days 1 and 5 (acute infection phase) and days 14 and 30 (chronic infection phase) post-challenge. The results showed the ability of water-in-oil emulsion combined with inactivated 5 × 10(5) tachyzoites to induce protection against neosporosis during the chronic stage, limiting parasite multiplication in the brain. Aluminum hydroxide-ginseng extract and inactivated tachyzoites reduced the number of parasites circulating in the blood during acute phase but failed to limit the establishment of chronic infection. On the other hand, a dose-effect was observed in groups vaccinated with aluminum hydroxide-ginseng extract in which the lesion severity increased as the inactivated tachyzoite dose. This study demonstrates that efficacy can significantly vary depending on the adjuvant, the dose of antigen and the phase of N. caninum infection in which the vaccine is tested.
Subject(s)
Adjuvants, Immunologic/administration & dosage , Antigens, Protozoan/administration & dosage , Coccidiosis/veterinary , Neospora/immunology , Protozoan Vaccines/immunology , Adjuvants, Immunologic/standards , Animals , Antibodies, Protozoan/blood , Antigens, Protozoan/immunology , Brain/parasitology , Brain/pathology , Coccidiosis/immunology , Coccidiosis/prevention & control , DNA, Protozoan/analysis , DNA, Protozoan/isolation & purification , Dose-Response Relationship, Immunologic , Female , Immunoglobulin G/blood , Injections, Subcutaneous/veterinary , Lung/parasitology , Mice , Mice, Inbred BALB C , Models, Animal , Neospora/genetics , Neospora/pathogenicity , Parasitemia/parasitology , Parasitemia/veterinary , Protozoan Vaccines/administration & dosage , Time Factors , Vaccination/veterinary , Vaccines, Inactivated/administration & dosage , Vaccines, Inactivated/immunologyABSTRACT
The objective of the present study was to analyze an extracellular component from Staphylococcus aureus (S. aureus), which we refer to as slime associated antigenic complex (SAAC), and to investigate the role of SAAC-specific antibody production in protection from S. aureus bovine mastitis. Twelve primiparous gestating cows were randomly assigned to one of the three groups: Group 1 was vaccinated with a S. aureus bacterin with very limited SAAC content; Group 2 received a S. aureus bacterin with high SAAC content and Group 3 served as unvaccinated controls. Animals were vaccinated at 45 days before the expected parturition date and revaccinated 35 days later. All groups were challenged by intramammary infusion with a virulent heterologous strain of S. aureus 23 days after calving. Antibody response against SAAC in serum and in milk, general clinical signs, mastitis score, somatic cell count (SCC) and count of S. aureus in milk were evaluated before and after challenge. Immunization with a high SAAC content in the S. aureus bacterin (Group 2) significantly enhanced antibody titers against SAAC (in serum and milk) and reduced the S. aureus concentration in milk during the post-challenge period compared to Group 1 and Group 3. Moreover, a significant negative correlation was observed between SAAC antibody production on the day of the challenge and the S. aureus count in milk by 1 day after challenge. However, there was no evidence of a difference between vaccinated and control groups with regard to clinical signs of mastitis following the challenge. Nevertheless, the SAAC antibody concentration on the day of the challenge negatively correlated with the mastitis score in quarters infected with S. aureus at 2 days post-challenge. These results indicate that the vaccines did not prevent S. aureus intramammary infection (IMI) after the experimental challenge, but immunization with a S. aureus bacterin with high SAAC content was able to reduce S. aureus multiplication in the mammary gland after challenge and suggests that the SAAC-specific antibody response could be involved in the protection against S. aureus intramammary infection. Although further studies should be performed to confirm the efficacy (under experimental conditions and in field trials), we propose bacterins from strong biofilm-producing bacteria and with high SAAC content, rather than with limited SAAC content, as a cost-efficient vaccine design against S. aureus bovine mastitis.