23 April 2023 Episode 330 The future of Flu vaccines: mRNA, protein or vector?

Sun, 04/23/2023 - 17:38

Episode 330: The future of flu vaccines: mRNA, protein or vector?

Dear colleagues,

The rapid and successful development of COVID vaccines, based on “new” principles, has raised expectations for better flu vaccines.  Par 1 is  a short reminder, copy-pasted from Ep 313. In Par 2 we will first evaluate recent progress in vaccines against seasonal flu.  In Par 3 some recent reports on bird flu will be discussed and in Par 4, we will reflect on the prospects for a “universal flu vaccine”. 

As you will see, the mRNA or recombinant protein technology may contribute to a better, even potentially “universal” flu vaccine, but there are caveats, especially in “naïve” individuals.


Par 1 Introductory reminder on the flu basics



Influenza A virus (IAV) genome organization and virion structure.


(A) Genome organization: 8 single-stranded, negative-sense, viral (v)RNA segments PB2, PB1, PA, HA, NP, NA, M and NS of IAV


(Black boxes at the end of each of the vRNAs indicate the 3’ and 5’ non-coding regions (NCR).

Hatched boxes = the packaging signals present at the 3’ and 5’ ends of each of the vRNAs that are responsible for efficient encapsidation into nascent virions.

Numbers represent nucleotide lengths for each of the NCR and packaging signals; )


(B) Virion structure:

  • IAV is surrounded by a lipid bilayer containing the two viral glycoproteins hemagglutinin (HA), responsible  for binding to sialic acid-containing receptors; and neuraminidase (NA), responsible for viral release from infected cells.
  • Also in the virion membrane is the ion channel matrix 2 (M2) protein.
  • Under the viral lipid bilayer is a protein layer composed of the inner surface envelop matrix 1 (M1) protein, which plays a role in virion assembly and budding; and the nuclear export protein (NEP) involved in the nuclear export of the viral ribonucleoprotein (vRNP) complexes.
  • Underneath is the core made of the eight vRNA segments that are encapsidated by the viral nucleoprotein (NP).
  • Associated with each vRNP a complex is the viral RNA-dependent RNA polymerase (RdRp) complex made of the three polymerase subunits PB2, PB1 and PA that, together with the viral NP are the minimal components required for viral replication and transcription.




Fig. 2 | Influenza pandemics. In the past 100 years, four pandemics of human influenza have occurred, with the 1918 pandemic caused by an influenza A H1N1 virus being the most devastating, as it was associated with >40 million deaths.

Influenza A H2N2, H3N2 and H1N1 viruses caused the 1957 , 1968 and 2009 pandemics, respectively. In 1977 , influenza A H1N1 restarted circulation in humans without causing a pandemic, as the strain was similar to that which preceded the 1957 influenza A H2N2 pandemic. By contrast, the 2009 pandemic influenza A H1N1 virus was antigenically very different to the previous seasonal influenza A H1N1 viruses and replaced them as the circulating influenza A H1N1 strain.

Examples of the spreading of human influenza A viruses in the world are shown for pandemic 1918 and 1957 viruses and for seasonal H3N2 viruses18. For pandemic virus outbreaks, the arrows indicate the first and second waves of transmission.

For seasonal influenza A H3N2 spread, the arrows indicate the seeding hierarchy of seasonal influenza A(H3N2) viruses over a 5-year period, starting from a network of major cities in east and southeast Asia; the hierarchy within the city network is unknown. Seasonal influenza B viruses (not shown) are co- circulating in humans with influenza A viruses









Par 2 Recent developments in seasonal vaccines

Ep 330-1: As expected both Moderna and Pfizer-BioNTech are rushing to get results from phase 3 trials since the Autumn of 2022 comparing the “traditional” quadrivalent inactivated vaccine, with mRNA expressing hemagglutinin from corresponding 2 Influenza A and 2 Influenza B suspected seasonal strains.   

While no results were announced yet on Pfizer vaccine, interim (unpublished results by Moderna mRNA-1010 are called “mixed”:

mRNA-1010 demonstrated geometric mean titer ratios consistent with superiority against both influenza A strains (A/H1N1, A/H3N2) and consistent with non-inferiority against both influenza B strains (B/Victoria, B/Yamagata) relative to the licensed (inactivated) comparator.    

mRNA-1010 did not accrue sufficient cases at the interim efficacy analysis to declare early success in the Phase 3 Northern Hemisphere efficacy trial, and the independent DSMB recommended continuation of efficacy follow-up.


Ep 330-2: Novavax has published more convincing results with NanoFlu™, its recombinant protein quadrivalent seasonal influenza vaccine candidate with Matrix-M™ adjuvant:


  1. Press release: Sept 2021:
  • Enhanced wild-type hemagglutination-inhibiting antibody responses against homologous strains (22-66% increased) and six heterologous A/H3N2strains (34-46% increased) as compared to Fluzone Quadrivalent Inactivated comparator
  • Potent induction of polyfunctional antigen-specific CD4+ T-cells against A/H3N2and B/Victoria strains, with a 126–189% increase in various post-vaccination cell-mediated immunity markers as compared to Fluzone Quadrivalent


  1. Vivek Shinde Lancet Infect Dis 2022 : phase 3 in  community based 65+ adults:  1333 in the qNIV (= quadrivalent Nanoparticle Influenza Vaccine) group and 1319 in the IIV4 (quadrivalent Inactivated Influenza) group


Non-inferiority of hemagglutinin-inhibiting antibodies against homologous viruses (represented in the vaccine)




Non-inferiority against heterologous viruses



GMTR=geometric mean titre ratio. IIV4=quadrivalent inactivated influenza vaccine. LLoQ=lower limit of quantitation. qNIV=quadrivalent nanoparticle influenza vaccine. SCR=seroconversion rate.



Clearly enhanced CD4 T cell mediated immunity with Nano-Flu





Ep 330-3: NasdDaq stock market: Moderna's pain translates to Novavax's gain.



Ep 330-4: Sigrid Gouma J Virol Jan 2023: Nucleoside-Modified mRNA-Based Influenza Vaccines

Circumvent Problems Associated with H3N2 Vaccine Strain Egg Adaptation in mice


This paper shows that mRNA has 2 advantages over traditional inactivated vaccines:

  1. The original Hemagglutinin sequence can be used (avoiding the mutation, associated with egg-adaptation: Ab elicited with mRNA of WT (3c2) HA showed higher neutralization than after immunization with either mRNA of egg-adapted HA or traditional vaccine






  1. After challenge, both WT HA mRNA and egg-adapted HA mRNA provide better protection than inactivated vaccine




This is tentatively explained by the observation that both mRNA vaccines elicit much higher Ab against the more conserved “stalk” or “stem” part of HA than the inactivated vaccine




Conclusions from Par 2 (novel seasonal flu vaccines):


  • Messenger RNA of hemagglutinin could be more efficacious than inactivated virus, because it circumvents the egg-culture induced mutations and it induces T cell responses. 
  • Recombinant protein vaccine could have the same advantage, provided a potent Th1 adjuvant is added.  
  • The first results of the Moderna mRNA and the Novavax protein seem more promising for the latter (but a formal comparison has not been done).  



Par 3: More on the threat of  Bird Flu and how to face it


For a thorough introduction see Ep 319 and 320


As a reminder:



The H5 and H7 are highly pathogenic and the H9 is a low pathogenicity bird flu types. 

They belong to different clades: H5 is within H1 clade; H7 within H7 clade and H9 within H9 clade.



Nachbagauer Nat Med 2021 https://doi.org/10.1038/s41591-020-1118-7

There have been two major spill-over epidemics in 1997 with H5N1 and in 2013 H7 N9


This chronology demonstrates that it is only a matter of time before a future pandemic will occur.

It can be emerged by the accumulation of mutations or genetic reassortment.

Amongst the High Pathogenic Avian Influenza H5Nx and H7N9 are perceived to pose the highest danger.


At present most focus is on the H5N1 virus bearing the HA gene



The eight bars represent the eight gene segments (from top to bottom: PB2, PB1, PA, HA, NP, NA, M, and NS


As can be seen, this AIV got its H5 and M segment from Russia; its NA, PA and NS from two different Belgian H1N1 strains, while the PB1, PB2 and NP originate from a Mongolian and a Chinese H8N3 strains


Ep 330-5: Kai Kupfersmidt in Science 6 April: From bad to worse

Discussion on the changes that would be required to transform this bird flu into a human flu:

  1. Polymerase PB2: E627K (glutamic acidto lysine): has already been observed in H5N1 in infected foxes in the NL and a, infected New England seal.
  2. Hemagglutinin: Q226L (glutamine to leucin) and G228S (glycine to serine)
  3. Nucleoprotein must mutate into a form that the human MxA cannot detect



Reassuring conclusion? 

It has one of the highest barriers to becoming a pandemic virus of any avian influenza virus, Peacock says. “It’s really wrong in so many ways.  But obviously it only has to get the right combination of mutations once to do to jump.”

Ep 330-5: Jon Cohen Science 6 April 2023 discusses the slowly changing attitude towards vaccination of poultry.


The US and EU presently still do not vaccinate fearing that vaccinated poultry could still be infected, which could lead to further mutations and transmission, probably even to humans.  Therefore meat (even frozen) or eggs from vaccinated chickens is prohibited in US and EU.


Between January 2005 and 2022, more than 8500 H5 outbreaks led to the culling of nearly 400 million birds, with another 30 million sacrificed to fight more than 100 outbreaks of H7s.


Chen Hualan (Harbin Vet School):China has now about 10 years of experience with H5/H7 bivalent inactivated virus vaccination.

  • 70% of the chickens they sampled had antibodies from the H5N1 or the H5/H7 bivalent vaccines.
  • With regard to H5: Even though the globally circulating H5 viruses have been detected in many species of wild birds and occasionally in ducks or geese in recent years, they have never caused problems on routinely vaccinated poultry farms in China.
  • With regard to H7: they found the prevalence of the virus dropped by 93.3% , and the spread of H7N9 to humans stopped altogether. No human case of H7N9 has been reported since 2019.


David Swayne (US Dep Agriculture):

  • Vaccinated birds can still be infected with very high doses of live virus, but it is unlikely that they could become infected when exposed to more realistic doses.
  • No evidence exists that frozen meat can transmit infl uenza viruses. “Chances are low or negligible”


The European Union in May 2022 conceded that vaccinating might make sense and is now testing candidates against At an October 2022 meeting in Paris, organized under the aegis of the International Alliance for Biological Standardization, participants called for “removing unnecessary barriers” for vaccination to control HPAI.


In the US, the resistance against vaccination is still high, but “the unprecedented H5N1 outbreak in the United States has altered the conversation. Vaccination is discussed much more widely now, and there are some people who are pushing for it,”

In a statement to Science, USDA said if the vaccines work, which the agency should know by June, it will look for manufacturers to produce them. (It will also consider data from European labs now testing vaccines against,Spackman says.) Then, “there are 20 discrete stages to complete” before companies can submit data for regulatory approval, the statement says. It typically takes 2.5 to 3 years to complete that process, the agency said, but “in emergency situations manufacturers may expedite development, resulting in a shortened timeframe to licensure.”


Clearly, the attitude is changing, as scientific evidence doesn’t support the ban on vaccination and the threat by the present H5N1 bird fly pandemic is very real.



Ep 330-6: Shengkui Xu Vaccine March 2023: A new H9 mRNA-liponanoparticle vaccine elicits robust protective immunity in chickens.





The hemagglutinin-inhibition titers of low dose mRNA is lower than that after inactivated vaccine.




T cell immunity (IFN-g ELISPOT of splenocytes in C or blood in D) after mRNA is not inferior (rather superior) to inactivated vaccine.  




Protection against infection is evident and certainly not inferior to inactivated vaccine.


Conclusion from Par 3 (Bird Flu)

  • The presently circulating H5N1 is a dangerous virus, but mutations in several viral proteins would be required to transform it into a dangerous respiratory pathogen for humans
  • The EU and US, who have always been opposed to poultry vaccination, may change their attitude in view of the danger of H5N1 for the poultry industry and the favorable experience with poultry vaccination in China.
  • mRNA vaccination could offer an alternative to inactivated vaccines in poultry as well.


Par 4 Attempts towards a “Universal Flu Vaccine”




Antibodies against HA and NA, induced by inactivated Flu, are often poorly protective against infection, because of the variability of these surface proteins:  potential mismatch with actually circulating seasonal strains.  


Two possible solutions:

  • Inclusion of more conserved internal parts of Influenza (Matrix, Nucleoprotein etc) and counting on T cell immunity, especially to protect against severe disease.  
  • Inclusion of many different HA antigens.


Ep 330-7A: Thomas Evans Lancet Infect Dis 2022:  Efficacy and safety of a universal influenza A vaccine (MVA-NP+M1 = Modified Vaccinia Ankara, carrying the nucleoprotein and the Matrix protein) in human adults when given after seasonal inactivated quadrivalent influenza vaccine immunization.


No difference in incidence of laboratory-confirmed Flu



Despite temporary increase in T cell immunity




Trial was stopped for futility


Ep 330-7B: Valkenburgh Comment


The influenza season of this efficacy trial was potentially mild in terms of case severity, despite a heavy season in terms of case numbers, and the seasonal vaccine was relatively well matched for the circulating H3N2 strain.


Targeting mucosal immunity might be more important.



Ep 330-8: Koen van de Ven Science Feb 2023: A T cell–inducing nucleoside-modified mRNA vaccine that encodes the conserved nucleoprotein (NP), matrix protein 1 (M1), and polymerase basic protein 1 (PB1) of an H1N1 virus protects “experienced”, but not “naïve” ferrets, while inducing strong T cell immunity in both naïve and experienced.


To mimic the human situation, we applied the mRNA vaccine

  • as a prime-boost regimen in naïve ferrets (mimicking young children): two times mRNA day 0 and day 42
  • and as a booster in influenza-experienced ferrets (mimicking adults).  Influenza experience = intranasal infection with H1N1, followed by a booster on day 42


Protection against an heterologous avian Flu (H7N9) challenge?


Fig. 4. Boosting of existing immunity increases protection against H7N9 influenza virus challenge




(A) Study layout depicting the H7N9 influenza virus challenge after different prime-boost regimens. Ferrets were challenged intratracheally with 106 TCID50 /Anhui/1/2013 (H7N9) influenza virus at 71 or 72 dpp, which equals 0 dpi. At 5 dpi, animals were euthanized and tissue pathology and viral loads were assessed.

(B) Decrease in body weight from 0 to 5 dpi. Body weight is depicted relative to body weight (%) on the day of challenge.

(C) Clinical scoring for parameter activity and breathing as detailed in Materials and Methods. Ferrets reaching a combined score of 4 have reached the human end points and were euthanized



  • Influenza-experienced ferrets are well protected against the challenge.
  • The “naïve” ferrets in cage 1 were NOT protected and had to be euthanized, while those in the second cage showed some protection with regard to body weight loss, but not with regard to symptoms or other pathological changes (not shown here, but see Fig 4 C-F in the paper)



Fig 3: mRNA vaccination elicits strong IFN-g responses in CD8 T cells > CD4 T cells in various compartments



T cell responses are most pronounced in H1N1 infected + mRNA vaccinated ‘experienced”’ group > dually mRNA vaccinated “naïve” group > dually infected (H1N1 + N3N2) group > single infected (H1N1) group > placebo group.


No explanation found why the “naïve” (2 X mRNA) animals in cage 1 and cage 2 behaved so differently in terms of protection.



Ep 330-9: Claudia Arevalho Science Nov 2022: A multivalent nucleoside-modified mRNA vaccine against all known influenza virus HA subtypes protects mice and ferrets against matched and mismatched challenge.


Fig. 1. The 20-HA mRNA-LNP vaccine elicits long-lived antibody responses that react to all 20 Has in mice.



Mice were simultaneously vaccinated intramuscularly (i.m.) with 20 different HA mRNA-LNPs (a combined total dose of 50 mg of mRNA-LNP, including 2.5 mg of each individual HA mRNA-LNP from Influenza A group 1 and 2 as well as the 2 Influenza B Victoria and Yamagata).

Evaluated on day 28 and day 118.  AUC, area under the curve.


Fig. 3. The 20-HA mRNA-LNP vaccine protects mice from challenge with antigenically matched and mismatched distinct H1N1 strains.




Mice were vaccinated with mRNA-LNPs encoding H1 (blue), H3 (red), IBV (Influenza B Virus = gray), luciferase (Luc) (green), or 20 HAs (purple). 28 days later, they were infected intranasally (i.n.) with A/California/7/2009 [5 median lethal doses (LD50)] or A/Puerto Rico/8/1934 H1N1 (2 LD50) influenza virus.

(A Weight loss:  (B), clinical scores  and (C) survival (C) were monitored for 14 days



Fig. 4. 20-HA mRNA-LNP vaccination protects ferrets from challenge with an antigenically distinct H1N1 strain.




Ferrets were primed with 60 mg of the 20-HA mRNA-LNP vaccine (3 mg of each HA mRNA-LNP) and were then boosted with the same vaccine dose 28 days later.

  1. Sera were collected 28 days after the first and second vaccinations, and antibody reactivities to different HAs were quantified using ELISAs coated with recombinant proteins. 


Twenty-eight days after the second vaccination, ferrets were infected i.n. with 106 TCID50 of A/Ruddy turnstone/Delaware/300/2009 H1N1 influenza virus. As a control, unvaccinated animals were also infected with the virus.

  1. Weight loss; (C) survival, and (D) signs of disease (D) were monitored for 14 days after infection




  • A booster of an inactivated vaccine with MVA expressing nucleoprotein and matrix protein offers no clinical benefit over inactivated seasonal flu vaccine alone in a real world trial in humans.
  • An mRNA expressing NP, M1 and PB1 from H1N1 protects “influenza-experienced”, but not “naïve” ferrets against severe disease after challenge with a bird flu (H7N9) virus
  • A 20-valent hemagglutinin mRNA vaccine protects both mice and ferrets against severe disease after heterologous challenge.  


Best wishes,