The length of ribosomal binding site spacer sequence controls the production yield for intracellular and secreted proteins by Bacillus subtilis

BackgroundBacillus subtilis is widely used for the industrial production of recombinant proteins, mainly due to its high secretion capacity, but higher production yields can be achieved only if bottlenecks are removed. To this end, a crucial process is translation initiation which takes place at the ribosome binding site enclosing the Shine Dalgarno sequence, the start codon of the target gene and a short spacer sequence in between. Here, we have studied the effects of varying spacer sequence lengths in vivo on the production yield of different intra- and extracellular proteins.ResultsThe shuttle vector pBSMul1 containing the strong constitutive promoter PHpaII and the optimal Shine Dalgarno sequence TAAGGAGG was used as a template to construct a series of vectors with spacer lengths varying from 4 to 12 adenosines. For the intracellular proteins GFPmut3 and β-glucuronidase, an increase of spacer lengths from 4 to 7–9 nucleotides resulted in a gradual increase of product yields up to 27-fold reaching a plateau for even longer spacers. The production of secreted proteins was tested with cutinase Cut and swollenin EXLX1 which were N-terminally fused to one of the Sec-dependent signal peptides SPPel, SPEpr or SPBsn. Again, longer spacer sequences resulted in up to tenfold increased yields of extracellular proteins. Fusions with signal peptides SPPel or SPBsn revealed the highest production yields with spacers of 7–10nt length. Remarkably, fusions with SPEpr resulted in a twofold lower production yield with 6 or 7nt spacers reaching a maximum with 10–12nt spacers. This pattern was observed for both secreted proteins fused to SPEpr indicating a dominant role also of the nucleotide sequence encoding the respective signal peptide for translation initiation. This conclusion was corroborated by RT qPCR revealing only slightly different amounts of transcript. Also, the effect of a putative alternative translation initiation site could be ruled out.ConclusionOur results confirm the importance of the 5′ end sequence of a target gene for translation initiation. Optimizing production yields thus may require screenings for optimal spacer sequence lengths. In case of secreted proteins, the 5′ sequence encoding the signal peptide for Sec-depended secretion should also be considered

The fourth vaccination with a non-SARS-CoV-2 variant adapted vaccine fails to increase the breadth of the humoral immune response

Abstract Escape mutations in the spike protein of SARS-CoV-2 are a major reason for Omicron breakthrough infections. After basal vaccination only very low titers of Omicron neutralizing antibodies are present. However, booster vaccinations induce higher titers against the Omicron variant. The neutralization of the Delta and Omicron variants by sera obtained 6 months after 3rd vaccination and 2 weeks or 6 months after 4th vaccination with a monovalent RNA vaccine (Spikevax) was analyzed. It was observed for the Omicron variant that 6 months after the fourth vaccination, the titer returns to the same very low neutralizing capacity as 6 months after the third vaccination. The Delta variant neutralizing capacity wanes with a comparable kinetic although the titers are higher as compared to the Omicron variant. This indicates that the fourth vaccination with a monovalent vaccine based on the ancestral isolate neither affects the kinetic of the waning nor the breadth of the humoral response

Comirnaty-Elicited and Convalescent Sera Recognize Different Spike Epitopes

Many of the approved SARS-CoV-2 vaccines are based on a stabilized variant of the spike protein. This raises the question of whether the immune response against the stabilized spike is identical to the immune response that is elicited by the native spike in the case of a SARS-CoV-2 infection. Using a peptide array-based approach, we analysed the binding of antibodies from Comirnaty-elicited, convalescent, and control sera to the peptides covering the spike protein. A total of 37 linear epitopes were identified. A total of 26 of these epitopes were almost exclusively recognized by the convalescent sera. Mapping these epitopes to the spike structures revealed that most of these 26 epitopes are masked in the pre-fusion structure. In particular, in the conserved central helix, three epitopes that are only exposed in the post-fusion conformation were identified. This indicates a higher spike-specific antibody diversity in convalescent sera. These differences could be relevant for the breadth of spike-specific immune response

The SARS-CoV-2 Variant Omicron Is Able to Escape Vaccine-Induced Humoral Immune Responses, but Is Counteracted by Booster Vaccination

The SARS-CoV-2 variant Omicron has spread world-wide and is responsible for rapid increases in infections, including in populations with high vaccination rates. Here, we analysed in the sera of vaccinated individuals the antibody binding to the receptor-binding domain (RBD) of the spike protein and the neutralization of wild-type (WT), Delta (B.1.617.2), and Omicron (B.1.1.529; BA.1) pseudotyped vectors. Although sera from individuals immunized with vector vaccines (Vaxzevria; AZ and COVID-19 Janssen, Ad26.COV2.S; J&J) were able to bind and neutralize WT and Delta, they showed only background levels towards Omicron. In contrast, mRNA (Comirnaty; BNT) or heterologous (AZ/BNT) vaccines induced weak, but detectable responses against Omicron. While RBD-binding antibody levels decreased significantly six months after full vaccination, the SARS-CoV-2 RBD-directed avidity remained constant. However, this still coincided with a significant decrease in neutralization activity against all variants. A third booster vaccination with BNT significantly increased the humoral immune responses against all tested variants, including Omicron. In conclusion, only vaccination schedules that included at least one dose of mRNA vaccine and especially an mRNA booster vaccination induced sufficient antibody levels with neutralization capacity against multiple variants, including Omicron

Quantitative and Qualitative Difference in Antibody Response against Omicron and Ancestral SARS-CoV-2 after Third and Fourth Vaccination

Waning immunity against SARS-CoV-2 and the emergence of variants, especially of the most distant variant, Omicron, affect titers of neutralizing antibodies in the sera of vaccinated individuals. Thus, two vaccinations with the mRNA vaccine BNT162b fail to induce neutralizing antibodies against the Omicron variant. A first booster vaccination increases Omicron-RBD-binding IgG and IgA and neutralizing capacity. In comparison, the Wuhan isolate titers of the Omicron variant binding antibodies are 8.5 lower. After a third vaccination, induction of Omicron-RBD- and Wuhan-RBD-binding antibodies follows the same kinetic. Five to six months after the third vaccination, there are still Omicron-RBD-binding antibodies detectable, but 35.9 percent of the analyzed sera fail to neutralize the Omicron variant, while all sera efficiently neutralize the Delta isolate. In the case of the Wuhan-RBD, a significantly larger number of stable antigen–antibody complexes is formed than in Omicron-RBD. A fourth vaccination with mRNA-1273 temporarily restores levels of Omicron-, Delta- and Wuhan-specific antibodies. Comparing different booster strategies revealed that the breadth of the immune response is not affected by the vaccination regimen. Taken together, these data indicate that booster vaccinations (third and fourth dose) increase the breadth of the immune response, but there is a qualitative difference of antibodies with respect to the stability of antigen–antibody complexes and persistence of antibody titers

Analysis of BNT162b2- and CVnCoV-elicited sera and of convalescent sera toward SARS-CoV-2 viruses

BACKGROUND: The mRNA vaccine BNT162b2 (Comirnaty, BioNTech/Pfizer) and the vaccine candidate CVnCoV (Curevac) each encode a stabilized spike protein of SARS-CoV2 as antigen but differ with respect to the nature of the mRNA (modified versus unmodified nucleotides) and the mRNA amount (30 μg versus 12 μg RNA). This study characterizes antisera elicited by these two vaccines in comparison to convalescent sera. METHODS: Sera from BNT162b2 vaccinated healthcare workers, and sera from participants of a phase I trial vaccinated with 2, 4, 6, 8, or 12 μg CVnCoV and convalescent sera from hospitalized patients were analyzed by ELISA, neutralization tests, surface plasmon resonance (SPR), and peptide arrays. RESULTS: BNT162b2-elicited sera and convalescent sera have a higher titer of spike-RBD-specific antibodies and neutralizing antibodies as compared to the CVnCoV-elicited sera. For all analyzed sera a reduction in binding and neutralizing antibodies was found for the lineage B.1.351 variant of concern. SPR analyses revealed that the CVnCoV-elicited sera have a lower fraction of slow-dissociating antibodies. Accordingly, the CVnCoV sera almost fail to compete with the spike-ACE2 interaction. The significance of common VOC mutations K417N, E484K, or N501Y focused on linear epitopes was analyzed using a peptide array approach. The peptide arrays showed a strong difference between convalescent sera and vaccine-elicited sera. Specifically, the linear epitope at position N501 was affected by the mutation and elucidates the escape of viral variants to antibodies against this linear epitope. CONCLUSION: These data reveal differences in titer, neutralizing capacity, and affinity of the antibodies between BNT162b2- and CVnCoV-elicited sera, which could contribute to the apparent differences in vaccine efficacy

Comparative Investigation of Methods for Analysis of SARS-CoV-2-Spike-Specific Antisera

In light of an increasing number of vaccinated and convalescent individuals, there is a major need for the development of robust methods for the quantification of neutralizing antibodies; although, a defined correlate of protection is still missing. Sera from hospitalized COVID-19 patients suffering or not suffering from acute respiratory distress syndrome (ARDS) were comparatively analyzed by plaque reduction neutralization test (PRNT) and pseudotype-based neutralization assays to quantify their neutralizing capacity. The two neutralization assays showed comparable data. In case of the non-ARDS sera, there was a distinct correlation between the data from the neutralization assays on the one hand, and enzyme-linked immune sorbent assay (ELISA), as well as biophysical analyses, on the other hand. As such, surface plasmon resonance (SPR)-based assays for quantification of binding antibodies or analysis of the stability of the antigen–antibody interaction and inhibition of syncytium formation, determined by cell fusion assays, were performed. In the case of ARDS sera, which are characterized by a significantly higher fraction of RBD-binding IgA antibodies, there is a clear correlation between the neutralization assays and the ELISA data. In contrast to this, a less clear correlation between the biophysical analyses on the one hand and ELISAs and neutralization assays on the other hand was observed, which might be explained by the heterogeneity of the antibodies. To conclude, for less complex immune sera—as in cases of non-ARDS sera—combinations of titer quantification by ELISA with inhibition of syncytium formation, SPR-based analysis of antibody binding, determination of the stability of the antigen–antibody complex, and competition of the RBD-ACE2 binding represent alternatives to the classic PRNT for analysis of the neutralizing potential of SARS-CoV-2-specific sera, without the requirement for a BSL3 facility