Broadly protective immunity against divergent influenza viruses by oral co-administration of Lactococcus lactis expressing nucleoprotein adjuvanted with cholera toxin B subunit in mice
Authors: Xiaojue Peng
Jiexiu Ouyang
Han Lei
Daxian Zhao
Huifeng Jiao
Journal: Microbial cell factories (2015)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: influenza vaccine; universal vaccine; cross-protection; influenza virus A; nucleoprotein
Affiliations: College of Medicine, Southwest Jiaotong University, Chengdu, 610031, Sichuan, China. ., Department of Biotechnology, College of Life Science, Nanchang University, Nanchang, 330031, Jiangxi, China. .

Abstract:  Background: Current influenza vaccines need to be annually reformulated to well match the predicated circulating strains. Thus, it is critical for developing a novel universal influenza vaccine that would be able to confer cross-protection against constantly emerging divergent influenza virus strains. Influenza virus A is a genus of the Orthomyxoviridae family of viruses. Influenza virus nucleoprotein (NP) is a structural protein which encapsidates the negative strand viral RNA, and anti-NP antibodies play role in cross-protective immunity. Lactococcus lactis (L. lactis) is an ideal vaccine delivery vehicle via oral administration route. However, L. lactis vectored vaccine exhibits poor immunogenicity without the use of mucosal adjuvant. To enhance the immunogenicity of L. lactis vectored vaccine, cholera toxin B (CTB) subunit, one of mucosal adjuvants, is a safe adjuvant for oral route, when combined with L. lactis vectored vaccine. In this study, we hypothesized that pNZ8008, a L. lactis expression plasmid, encoding NP antigen, would be able to elicit cross-protection with the use of CTB via oral administration route. Results: To construct L. lactis vectored vaccine, nucleoprotein (NP) gene of A/California/04/2009(H1N1) was sub-cloned into a L. lactis expression plasmid, pNZ8008. The expression of recombinant L. lactis/pNZ8008-NP was confirmed by Western blot, immunofluorescence assay and flow cytometric analysis. Further, immunogenicity of L. lactis/pNZ8008-NP alone or adjuvanted with cholera toxin B (CTB) subunit was evaluated in a mouse model via oral administration route. Antibodies responses were detected by ELISA. The result indicated that oral administration of L. lactis/pNZ8008-NP adjuvanted with CTB could elicit significant humoral and mucosal immune responses, as well as cellular immune response, compared with L. lactis/pNZ8008-NP alone. To further assess the cross-protective immunity of L. lactis/pNZ8008-NP adjuvanted with CTB, we used L. lactis/pNZ8110-pgsA-HA1 alone or adjuvanted with CTB as controls. Mice that received L. lactis/pNZ8008-NP adjuvanted with CTB were completely protected from homologous H1N1 virus and showed 80% protection against heterologous H3N2 or H5N1 virus, respectively. By contrast, L. lactis/pNZ8110-pgsA-HA1 adjuvanted with CTB also conferred 100% protection against H5N1 virus infection, but indicated no cross-protection against H1N1 or H5N1 virus challenge. As controls, mice vaccinated orally with L. lactis/pNZ8008-NP alone or L. lactis/pNZ8110-pgsA-HA1 alone could not survive. Conclusion: This study is the first to report the construction of recombinant L. lactis/pNZ8008-NP and investigate its immunogenicity with the use of CTB. Compared with L. lactis/pNZ8110-pgsA-HA1 adjuvanted with CTB, our data support 5 × 10(11) CFU of L. lactis/pNZ8008-NP adjuvanted with 1 µg of CTB is a better combination for universal influenza vaccines development that would provide cross-protective immunity against divergent influenza A viruses. 

Retracted: No

PubPeer comments:
 Brought to my attention by a reader. This paper is one of a set of six papers that all share the same authors, were all published in 2014-2015, and that all share very similar plots. No matter what animal species (mouse, chicken, or ferret) or number of animals per group (6-25), the body weight loss in unprotected animals was all very similar (around 25% loss at day 6), while the protected animals all show a dip in body weight around 6 days, followed by an increase. In addition, the error bars (standard deviations in all papers) are unexpectedly small for these types of measurements. Could the authors please provide some explanation for these similarities? The 6 papers are the following: * Han Lei et al. Virology 476 (2015) 189u2013195, DOI: 10.1016 j.virol.2014.12.017, https: pubpeer.com publications E932C6D86F69113141665259EB4C79 * Han Lei et al. Arch Virol (2015) 160:3011u20133019, DOI: 10.1007 s00705-015-2587-8, https: pubpeer.com publications 4E88CDED2683B085DE9D52B7AD7700 * Han Lei et al. BMC Veterinary Research (2015) 11:85, DOI: 10.1186 s12917-015-0399-4, https: pubpeer.com publications B407285D6C4C238DC25A4FA0F28991 * Han Lei et al. Virus Research 196 (2015) 56u201359, DOI: 10.1016 j.virusres.2014.11.009, https: pubpeer.com publications 889BA6B1AE96E94614521AA9583E87 * Han Lei et al. Microb Cell Fact (2015) 14:111, DOI: 10.1186 s12934-015-0287-4, https: pubpeer.com publications A13874959B0962999C5A01039B8D02 * Han Lei et al. Journal of Medical Virology (2014), DOI: 10.1002 jmv.23983, https: pubpeer.com publications BFBF1A36BD6B9C54611A9727DA0206 And here are the graphs: ![file] ![file] ![file] ![file] ![file] , 
 Dear Elisabeth M Bik, Thank you very much for your comments. On behalf of all authors, I would like to address your concerns. Virus challenge is a gold-standard method for evaluating immune protection of viral vaccine candidate. Animal mode will be monitored for survival and body weight change for 14 days at post challenge, the criteria of virus challenge is that negative controls would be died within 6-8 days and the endpoint is body weight loss exceeded 25% relative to the weight at the time of challenge inoculation. Therefore, in our studies, different animal models such as mice, chickens or ferrets adapted influenza viruses with different virus dose are chosen for virus challenge experiment for investigating different L. lactis based oral or intranasal vaccine. Unprotected animals that you mentioned are negative controls such as PBS group and L lactis containing empty plasmid group were died within 6-8 days at post animal adapted influenza virus in our publications. Meanwhile, the body losses of these negative controls exceeded 25% would be euthanasia at post challenge, because these negative controls did not produce protective immunity. In contrast, the body weight of animal model vaccinated with L lactis based vaccine would increase on days 6-8 at post challenge, because they could produce protective immunity supported by antibody responses and HI assay. Thus, days 6-8 is a critical time point for virus challenge. The error bars in our publications is unexpectedly small as you mentioned, because the values of standard deviations are derived from statistical software based on our original data at post challenge. Thank you very much for your interests. Please let us know if you have any questions. , 
 Dear Dr. Lei, thank you for your reply. Could you please elaborate on your writing here :  The error bars in our publications is unexpectedly small as you mentioned, because the values of standard deviations are derived from statistical software based on our original data at post challenge.  Standard deviations are not randomly made up by software, but they are based on the variation within the measured values. Are you saying you did not actually measure these values? Or, if you did, could you share with us those measured values please? , 
 Thank you very much for your interests. Please let us know if you have any questions. Dear Elisabeth M Bik, we would like to address your concern.  The error bars in our publications is unexpectedly small as you mentioned, because the values of standard deviations are derived from statistical software based on our original data at post challenge.  means that we input our original data to graphics software (Origin 75), and then we chose  statistical analysis button to obtain Mean value an SD value. Based on these Meanu00b1SD valure, we could draw figures via Origin 75 software. Therefore, we definitely measured Means and SD values based on origin data in our publication. Again, please let us know, if you have any questions. ,