Replicating bacterium-vectored vaccine expressing SARS-CoV-2 Membrane and Nucleocapsid proteins protects against severe COVID-19 disease in hamsters

An inexpensive readily manufactured COVID-19 vaccine that protects against severe disease is needed to combat the pandemic. We have employed the LVS ΔcapB vector platform, previously used successfully to generate potent vaccines against the Select Agents of tularemia, anthrax, plague, and melioidosis, to generate a COVID-19 vaccine. The LVS ΔcapB vector, a replicating intracellular bacterium, is a highly attenuated derivative of a tularemia vaccine (LVS) previously administered to millions of people. We generated vaccines expressing SARS-CoV-2 structural proteins and evaluated them for efficacy in the golden Syrian hamster, which develops severe COVID-19 disease. Hamsters immunized intradermally or intranasally with a vaccine co-expressing the Membrane (M) and Nucleocapsid (N) proteins, then challenged 5-weeks later with a high dose of SARS-CoV-2, were protected against severe weight loss and lung pathology and had reduced viral loads in the oropharynx and lungs. Protection by the vaccine, which induces murine N-specific interferon-gamma secreting T cells, was highly correlated with pre-challenge serum anti-N TH1-biased IgG. This potent vaccine against severe COVID-19 should be safe and easily manufactured, stored, and distributed, and given the high homology between MN proteins of SARS-CoV and SARS-CoV-2, has potential as a universal vaccine against the SARS subset of pandemic causing β-coronaviruses.

The ongoing pandemic of COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused over 50 million cases and 1.2 million deaths as of this writing 1 . A safe and potent vaccine that protects against severe COVID-19 disease is urgently needed to contain the pandemic. Ideally, such a vaccine would be safe, inexpensive, rapidly manufactured, and easily stored and distributed, so as to be available quickly to the entire world population.
Previously, our laboratory developed a versatile plug-and-play Single Vector Platform Vaccine against Select Agents and Emerging Pathogens wherein a single live multi-deletional attenuated Francisella tularensis subsp. holarctica vector, LVS ΔcapB, is used to express recombinant immunoprotective antigens of target pathogens 2,3 . The LVS ΔcapB vector was derived via mutagenesis from Live Vaccine Strain (LVS), a vaccine against tularemia originally developed in the Soviet Union via serial passage and subsequently further developed and tested in humans in the USA 4,5 . As with wild-type F. tularensis, LVS is ingested by host macrophages via looping phagocytosis, enters a phagosome, escapes the phagosome via a Type VI Secretion System, and multiplies in the cytoplasm [6][7][8] . While much more attenuated than LVS, the LVS ΔcapB vector retains its parent's capacity to invade and multiply in macrophages 9 . Using this platform technology, we have developed exceptionally safe and potent vaccines that protect against lethal respiratory challenge with the Tier 1 Select Agents of four diseases -tularemia, anthrax, plague, and melioidosis 2,3 . These vaccines induce balanced humoral (antibody/neutralizing antibody in the case of anthrax toxin) and cell-mediated immune responses (polyfunctional CD4+ and CD8+ T-cells) against key immunoprotective antigens of target pathogens 3 . We have now used this platform to develop a COVID-19 vaccine. SARS-CoV-2 has four structural proteins -the Spike (S) glycoprotein, Membrane (M), Envelope (E), and Nucleocapsid (N) proteins. Virtually all COVID-19 vaccines in development have focused on the S protein, which mediates virus entry into host cells via the Angiotensin Converting Enzyme 2 (ACE2) receptor 10,11 . These vaccines have been tested for efficacy most prominently in the rhesus macaque model of COVID-19. However, this is primarily a model of asymptomatic infection or mild disease, as animals typically do not develop either fever or weight loss; hence, vaccine efficacy in the rhesus macaque is quantitated primarily in terms of the vaccine's impact on viral load rather than on clinical symptoms. In contrast, the golden Syrian hamster develops severe COVID-19 disease, akin to that of hospitalized humans 12 , including substantial weight loss and quantifiable lung pathology.
Herein, we have employed the LVS ΔcapB vector platform to construct six COVID-19 vaccines expressing one or more of all four structural proteins of SARS-CoV-2 (S, SΔTM, S1, S2, S2E, and MN) and tested the vaccines for efficacy, administered intradermally (ID) or intranasally (IN), against a high dose SARS-CoV-2 respiratory challenge in hamsters. We show that the vaccine expressing the MN proteins, but not the vaccines expressing the S protein or its subunits in various configurations, is highly protective against severe COVID-19 disease including weight loss and lung pathology, and that protection is highly correlated with serum anti-N antibody levels.
Construction and verification of rLVS ΔcapB/SCoV2 vaccine candidates. We constructed six recombinant LVS ΔcapB vaccines (rLVS ΔcapB/SCoV2) expressing single, subunit or fusion proteins of four SARS-CoV-2 structural proteins: S 13 , E, M, and N (Fig. 1A). The S protein is synthesized as a single-chain inactive precursor of 1,273 residues with a signal peptide (residue 1-15) and processed by a furin-like host proteinase into the S1 subunit that binds to host receptor ACE2 10 and the S2 subunit that mediates the fusion of the viral and host cell membranes. S1 contains the host receptor binding domain (RBD) and S2 contains a transmembrane domain (TM) (Fig. 1B, top panel). We constructed rLVS ΔcapB/SCoV2 expressing S (stabilized) and, so as to express lower molecular weight constructs, SΔTM, S1, S2, and the fusion protein of S2 and E (S2E), and additionally, a vaccine expressing the fusion protein of M and N (MN) (Fig.   1B, bottom panels). A 3FLAG-tag was placed at the N-terminus of the S, SΔTM, S1, and MN proteins. The antigen expression cassette of the SARS-CoV-2 proteins was placed downstream of a strong F. tularensis promoter (Pbfr) and a Shine-Dalgarno sequence (Fig. 1B)  All six rLVS ΔcapB/SCoV2 vaccine candidates, abbreviated as S, SΔTM, S1, S2, S2E, and MN, expressed the recombinant proteins from bacterial lysates. As shown in Fig. 1C, three protein bands -a minor 75 kDa, a major 46 kDa, and a minor 30 kDa band -were detected from lysates of 4 individual clones of the MN vaccine candidate (Fig. 1C, lanes 3-6), but not from the lysate of the vaccine vector (lane 2) by Western blotting using guinea pig polyclonal antibody to SARS-CoV, which also detected the N and S protein of SARS-CoV (lanes 7 and 8, respectively).
The 75-, 46-, and 30-kDa protein bands represent the full-length MN, the N, and degradations of the MN protein. The S, SΔTM, S1, S2, and S2E proteins were also expressed by the rLVS ΔcapB/SCoV2 vaccines, as evidenced by Western blotting analysis using monoclonal antibody to FLAG to detect S, SΔTM, and S1 (each with an N-terminus FLAG tag) and polyclonal antibody to SARS-CoV to detect non-tagged S2 protein (Fig. S1, A-D). Of note, SΔTM and S1 ( Fig. S1B) were expressed more abundantly than the full-length S protein (Fig. S1A), possibly as a result of the removal of the TM domain and reduced size of the protein.  (Table S1A). All animals lost weight during the first 2 days after challenge; however, hamsters immunized with the MN vaccine, alone or in combination with the SΔTM or S1 vaccine, began to recover from the weight loss starting on Day 3, whereas shamimmunized animals continued to lose weight until euthanized on Day 7, by which time they had lost a mean of 8% of their total body weight. Hamsters immunized with the vector control continued to lose weight until Day 5 and then exhibited a small partial recovery, possibly reflecting a small beneficial non-specific immunologic effect as has been hypothesized for BCG and other vaccines. In contrast to hamsters immunized with the MN vaccine, hamsters immunized with the S, SΔTM, S1, S2, or S2E vaccines, administered ID or IN, were not protected against severe weight loss (Fig. 2B,  To evaluate viral replication in the lungs, we assayed cranial and caudal lungs for viral load on Day 3 post-challenge, which peaks at this time point in unvaccinated animals. Hamsters immunized ID with the MN vaccine, alone or in combination with the SΔTM or S1, showed significantly reduced viral loads in their cranial and caudal lungs compared with sham-or vectorimmunized animals (Fig. 4B, left panel). Hamsters immunized ID with the MN vaccines as a group showed a mean reduction of 0.8±0.1 log compared with Sham (P< 0.0001). In contrast, hamsters immunized ID with the S (S, SΔTM, S1, S2, S2E) protein vaccines did not show reduced viral loads in their cranial and caudal lungs (data not shown). Similar results were observed in hamsters immunized IN (Fig. 4B, right panel).

MN expressing vaccines induce antibody to N protein with a TH1 bias.
To assess antibody responses to SARS-CoV-2 proteins expressed by the vaccine, we analyzed antibodies to the RBD of the S protein and to the N protein (Fig. 5). As expected, sera from sham-and vectorimmunized hamsters lacked antibody to either antigen ( Fig. 5A-C). In contrast, sera from hamsters immunized once with the MN vaccine, alone or in combination with the SΔTM or S1 vaccine, showed high levels of N specific IgG, whether immunized ID or IN, at 3 weeks postimmunization ( Fig. 5A), which somewhat increased at Week 8, 5 weeks after the second immunization at Week 3 (Fig. 5B), displaying a TH1 type bias, with IgG2 dominating the response (Fig. 5C). Differences in serum anti-N IgG titers between hamsters immunized with the MN vaccine, alone or in combination with S protein vaccines, and sham-or vector-immunized hamsters were highly significant at both Week 3 and Week 8 (P<0.0001) (Fig. 5D).

Surprisingly, hamsters immunized with S protein vaccines did not show anti-RBD antibody at
Week 3 (Fig. 5A), nor SARS-CoV-2 neutralizing antibody at Week 8 (data not shown). In mice immunized at Weeks 0 and 3 with second generation vaccines expressing MN in combination with S1 or SΔTM, serum obtained at Week 4 showed anti-RBD antibody as well as anti-N antibody (Fig. S2). Anti-N IgG antibody displayed a TH1 type bias both in hamsters (Fig. 5C), where IgG2 dominated the IgG response, and in mice, where IgG2a dominated the IgG response (Fig. S2). This TH1 bias was also reflected by murine splenocyte secretion of IFN- in response to S and N peptides (Fig. S3).

Serum anti-N antibody correlates with protection in hamsters.
We assessed the correlation coefficient between serum anti-N IgG antibody just before challenge at Week 8 and lung (cranial + caudal) histopathological scores at Day 7 post-challenge by linear regression analysis. Anti-N antibody was highly and inversely correlated with histopathology score (R 2 = 0.9903, P< 0.0001) (Fig. 5E). This antibody, which does not neutralize SARS-CoV-2 (data not shown), likely is not itself protective but instead correlates with a protective T cell response such as that shown in Fig.   S3.

Discussion
We show that a replicating LVS ΔcapB-vectored COVID-19 vaccine, rLVS ΔcapB/SCoV2 MN, that expresses the SARS-CoV-2 M and N proteins, protects against COVID-19 disease in the demanding golden Syrian hamster model. The vaccine significantly protects against weight loss and severe lung pathology, the two major clinical endpoints measured, and significantly reduces viral titers in the oropharynx and lungs. The vaccine was protective after either ID or IN administration.
Surprisingly, of the six vaccines expressing one or more of the four SARS-CoV-2 structural proteins, only the vaccine expressing the MN proteins was protective. Such a vaccine has the potential to provide cross-protective immunity against the SARS subgroup of β-coronaviruses including potential future pandemic strains. While the S protein shows only 76% sequence identity between SARS-CoV and SARS-CoV-2, the M and N proteins each show 90% identity 14 . In an analysis of T-cell epitopes in humans recovered from COVID, the M and N antigens together accounted for 33% of the total CD4+ T cell response (21% and 11% for M and N, respectively) and 34% of the total CD8+ T cell response (12% and 22% for M and N, respectively), an amount exceeding the 27% and 26% CD4 and CD8 T cell responses, respectively, of the S protein 15 . Hence, the MN vaccine has potential for universal protection against this group of especially severe pandemic strains.
We evaluated our vaccines in the hamster model of SARS-CoV-2 infection because of its high similarity to serious human COVID-19 disease, which likely reflects at least in part the high genetic similarity of the hamster and human ACE2 receptor -S protein interface. A modelling of binding affinities showed that the hamster ACE2 has the highest binding affinity to SARS-CoV-2 S of all species studied with the exception of the human and rhesus macaque.
In our previous studies of vaccines utilizing the LVS ΔcapB vector platform, three immunization doses consistently yielded superior efficacy to two doses. Here, given the urgency for a COVID-19 vaccine and the desire to simplify the logistics of vaccine administration, we opted to test only two immunizations, while still maintaining a reasonably long immunization-challenge interval (5 weeks after the second immunization). Future studies will examine if three doses are superior to two and the longevity of immunoprotection. These last three advantages are particularly important with respect to making a COVID-19 vaccine available rapidly and cheaply to the entire world's population.
Safety is always a major consideration in vaccine development, especially so in the case of replicating vaccines. In our vaccine's favor, its much less attenuated parent (LVS) was already considered safe enough to justify extensive testing in humans, including recently, and it has demonstrated safety and immunogenicity 5,[23][24][25][26][27][28][29] . LVS has two major attenuating deletions and several minor ones 30 . As many as 60 million Russians were reportedly vaccinated against tularemia with the original LVS strain 31 , and over 5,000 laboratory workers in the United States have been vaccinated with the modern version of LVS by scarification 5 . Our further attenuation of LVS by introduction of the capB mutation reduced its virulence in mice by the IN route by >10,000-fold 9 . Hence, rLVS ΔcapB/SCoV2 MN and other LVS ΔcapB-vectored vaccines are anticipated to be exceedingly safe.
Correlates of protective immunity to COVID-19 are not well understood. Almost all of the vaccines in development are centered on generating immunity to the S protein -especially neutralizing antibody to this protein. However, neutralizing antibody alone may not be sufficient for full protection; vaccines generating strong neutralizing antibody responses against SARS-Co-V were not necessarily highly protective, especially in ferrets, which exhibit SARS disease more akin to that in humans 32,33 . T-cell responses may be as or more important. T cell responses were demonstrated to be required to protect against clinical disease in SARS-CoV challenged mice and adoptive transfer of SARS-CoV specific CD4 or CD8 T-cells into immunodeficient mice infected with SARS-CoV lead to rapid viral clearance and disease amelioration 34 .
Our S protein vaccines were ineffective, likely due to suboptimal S protein immunogenicity, reflected by the rapid decline of antibody titer in mice and the negligible antibody neutralization titers in hamsters just before challenge (data not shown). Possibly, enhanced or alternative expression of the S protein, for example display on the bacterial surface in addition to secretion, would improve immunogenicity, as reported for the S protein of SARS-CoV 35 . This would allow immune responses to the S protein to contribute to the already substantial protective efficacy provided by immune responses to the M and N proteins.
Our replicating bacterial vaccine expressing the M and N proteins has demonstrated safety and efficacy in an animal model of severe COVID-19 disease. If its safety and efficacy is reproduced in humans, the vaccine has potential to protect people from serious illness and death.
Considering the ease with which our vaccine can be manufactured, stored, and distributed, it has the potential to play a major role in curbing the COVID-19 pandemic, thereby saving thousands of lives and more rapidly restoring the world's battered economy.