Nucleoside-modified mRNA vaccination partially overcomes maternal antibody inhibition of de novo immune responses in mice - PubMed (original) (raw)
Nucleoside-modified mRNA vaccination partially overcomes maternal antibody inhibition of de novo immune responses in mice
Elinor Willis et al. Sci Transl Med. 2020.
Abstract
Maternal antibodies provide short-term protection to infants against many infections. However, they can inhibit de novo antibody responses in infants elicited by infections or vaccination, leading to increased long-term susceptibility to infectious diseases. Thus, there is a need to develop vaccines that are able to elicit protective immune responses in the presence of antigen-specific maternal antibodies. Here, we used a mouse model to demonstrate that influenza virus-specific maternal antibodies inhibited de novo antibody responses in mouse pups elicited by influenza virus infection or administration of conventional influenza vaccines. We found that a recently developed influenza vaccine, nucleoside-modified mRNA encapsulated in lipid nanoparticles (mRNA-LNP), partially overcame this inhibition by maternal antibodies. The mRNA-LNP influenza vaccine established long-lived germinal centers in the mouse pups and elicited stronger antibody responses than did a conventional influenza vaccine approved for use in humans. Vaccination with mRNA-LNP vaccines may offer a promising strategy for generating robust immune responses in infants in the presence of maternal antibodies.
Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
Conflict of interest statement
Competing interests: S.E.H. has received consulting fees from Sanofi Pasteur, Lumen, Novavax, and Merck. B.L.M. and Y.K.T. are employed by and own shares of Acuitas Therapeutics, a company involved in the development of mRNA-LNP therapeutics. D.W. is a coinventor on the following patents that describe the use of nucleoside-modified mRNA as a platform to deliver therapeutic proteins: patent no. 11/990,646 “RNA containing modified nucleosides and methods of use thereof,” patent no.14/776,525 “Purification and purity assessment of RNA molecules synthesized with modified nucleosides,” and patent no. 13/585,517 “RNA containing modified nucleosides and methods of use thereof.” D.W. and N.P. are coinventors on patents describing the use of modified mRNA in lipid nanoparticles as a vaccine platform: patent no. WO/2016/176330 “Nucleoside-modified RNA for inducing an adaptive immune response” and patent no. WO/2018/081638 “Nucleoside-modified RNA for inducing an adaptive immune response.” B.L.M. and Y.K.T. are coinventors on patents that describe lipid nanoparticles for delivery of nucleic acid therapeutics including mRNA: patent no. WO/2016/176330 “Nucleoside-modified RNA for inducing an adaptive immune response,” patent no. WO/2018/081638 “Nucleoside-modified RNA for inducing an adaptive immune response,” patent no. WO/2018/191719 “Lipid delivery of therapeutic agents to adipose tissue,” patent no. WO/2018/081480 “Lipid nanoparticle formulations,” patent no. WO/2018/078053 “Lipid nanoparticle mRNA vaccines,” and patent no. WO/2019/089828 “Lamellar lipid nanoparticles.”
Figures
Fig. 1.. matAbs protect mouse pups from influenza disease but inhibit de novo antibody responses.
(A) The experimental design is shown. (B and C) Serum was collected from mothers and mouse pups on the day of weaning, and influenza virus–specific antibody titers were measured by ELISA (B) or hemagglutination inhibition assay (C). Each point represents one litter (1 to 10 pups per litter; mean, 5.3) (B) or one mouse (C). In (B), maternal antibody titers are shown on the x axis, and pup titers are shown on the y axis; dotted line, 95% confidence interval (CI) (_R_2 = 0.58). (D) Serum was collected at the indicated time points from pups with influenza virus–specific matAbs, and influenza virus–specific ELISA titers were measured. One-phase decay (_R_2 > 0.97 for each mouse) was fitted to titer data; each line represents one mouse (n = 6 mice). (E and F) Seven-day-old mice with or without influenza virus–specific matAbs were intranasally inoculated with a 30 tissue culture infectious dose (TCID)50 of PR8 influenza virus. (E) Survival was measured over 14 days post-inoculation. Mouse groups were n = 3 (+matAbs) or n = 5 (−matAbs). P = 0.008, log-rank test. (F) Influenza virus titers in the lungs of pups were measured 2 days post-inoculation. Each point represents one mouse; n = 4 (+matAbs) or n = 5 (−matAbs) mice per group; P < 0.0001, two-tailed Welch’s t test. (G) Seven-day-old mice were intranasally inoculated with influenza virus or PBS in the presence or absence of matAbs. Serum influenza virus–specific IgG was measured by ELISA 125 days post-inoculation. n = 6 (black and orange), n = 8 (blue), or n = 9 (yellow) mice per group. Groups were compared using one-way ANOVA with Tukey’s post hoc test. (H) C57BL/6 mouse pups born to mothers not exposed to influenza virus were fostered by virus-exposed BALB/c mothers and then inoculated with 3 TCID50 of PR8 influenza virus or PBS intranasally at 7 days old. Serum influenza virus–specific IgG2a (maternal antibody) or IgG2c (de novo antibody) was measured by ELISA 14 days post-inoculation. n = 3 (orange and yellow) or n = 6 (blue) mice per group. Groups were compared using one-way ANOVA with Tukey’s post hoc test. Data in (C), (F), (G), and (H) are shown as means ±SD. Panels (D) to (G) show the results of one experiment that is representative of three independent biological replicates. Panel (H) shows results of one experiment that is representative of two independent biological replicates. ns, not significant.
Fig. 2.. matAbs inhibit antibody responses to conventional influenza vaccines.
(A) The experimental design is shown. (B and D) Mice (21 days old) with or without influenza virus–specific matAbs were inoculated with 10 TCID50 of PR8 influenza virus intranasally (B) or 1000 hemagglutination units of purified inactivated PR8 influenza virus intramuscularly (D) or PBS as a vehicle control. Serum influenza virus–specific antibody responses were measured over time. *P < 0.05 after comparison of serum titers from mice exposed to influenza virus versus PBS in the presence (blue *) or absence (orange *) of influenza virus–specific matAbs; one-way ANOVA with Tukey’s post hoc test at each time point. ns, not significant. (C and E) Mice inoculated in (B) and (D) were challenged at 189 or 194 days post-vaccination with 300 TCID50 of PR8 influenza virus intranasally, and weight loss was measured over 14 days. Data are shown as percentage of baseline weight (current weight divided by prechallenge weight). *P < 0.05 after comparison of percentage of baseline weight on each day for mice exposed to influenza virus versus PBS as infants in the presence (blue *) or absence (orange *) of influenza virus–specific matAbs; one-way ANOVA with Tukey’s post hoc test at each day. ns, not significant. In (E), one mouse survived in the blue group. n = 5 (blue), n = 6 (yellow), or n = 7 (orange and black) mice per group (B and C); n = 3 (black), n = 4 (blue and orange), or n = 11 (yellow) mice per group (D and E). Data in (B) to (E) are shown as means ± SD. Panels (B) and (C) show the results of one experiment that is representative of three independent biological replicates. Panels (D) and (E) show the results of one experiment that is representative of two independent biological replicates.
Fig. 3.. PR8 hemagglutinin mRNA-LNP vaccine elicits protective antibody responses in the presence of matAbs.
(A) Mice (21 days old) were vaccinated intramuscularly with 1 μg of nucleoside-modified PR8 hemagglutinin (HA) mRNA-LNP vaccine, and influenza virus–specific serum antibody responses were measured by ELISA. *P < 0.05 after comparison of serum titers from mice vaccinated with PR8 HA mRNA-LNP vaccine versus PBS in the presence (blue *) or absence (orange *) of influenza virus–specific matAbs; one-way ANOVA with Tukey’s post hoc test at each time point. (B) Mice in (A) were challenged at 189 days after vaccination with 300 TCID50 of PR8 influenza virus intranasally, and weight loss was measured over 14 days. Data are shown as percentage of baseline weight (current weight divided by prechallenge weight). *P < 0.05 after comparison of percentage of baseline weight on each day for mice vaccinated as infants with PR8 HA mRNA-LNP vaccine versus PBS in the presence (blue *) or absence (orange *) of influenza virus–specific matAbs; one-way ANOVA with Tukey’s post hoc test at each day. (A and B) n = 3 (+matAbs) or n = 4 (−matAbs) mice per group. (C) Serum was collected at 100+ days after vaccination with 1 μg of PR8 HA mRNA-LNP vaccine or PBS in the presence or absence of influenza virus–specific matAbs and was pooled. Pooled serum (500 μl) was intraperitoneally transferred to 6- to 8-week-old naïve mice, and 4 to 5 hours later, mice were intranasally challenged with 300 TCID50 of PR8 influenza virus. Weight loss was measured over 14 days. n = 4 mice per group. *P < 0.05 after comparison of percentage of baseline weight on each day for mice that received sera from mice vaccinated with PR8 HA mRNA-LNP vaccine or PBS as infants in the presence of influenza virus–specific matAbs; one-way ANOVA with Tukey’s post hoc test at each day. (D) Sera from mice vaccinated with 1 μg of PR8 HA mRNA-LNP vaccine in the presence or absence of influenza virus–specific matAbs and from naïve mice were collected 189 days post-vaccination. Sera were analyzed for influenza virus–specific IgG1 (left) or IgG2a (right). n = 8 (blue), n = 9 (orange), or n = 4 (black) mice per group. Serum titers of mice vaccinated with PR8 HA mRNA-LNP vaccine in the presence or absence of influenza virus–specific matAbs were compared using an unpaired two-tailed t test. In (D), each point represents one mouse. Data are shown as means ± SD. Panels (A) to (D) show data from one experiment that is representative of two independent biological replicates.
Fig. 4.. Cell-associated and secreted PR8 HA mRNA-LNP vaccines elicit similar antibody responses, and a low dose of mRNA-LNP vaccine overcomes matAb inhibition.
(A) The schematic shows PR8 influenza virus hemagglutinin (HA) mRNA constructs. mRNA expressing full-length PR8 HA produced cell-associated (C) HA. For some experiments, we used mRNA expressing secreted (S) HA. For this construct, the transmembrane (TM) and cytoplasmic domains were removed, and a trimerization domain was introduced. (B) Mice (21 days old) were vaccinated with cell-associated (C) or secreted (S) PR8 influenza virus HA mRNA-LNP vaccine or PBS (−) in the presence or absence of influenza virus–specific matAbs. Serum was collected 70 days post-vaccination, and influenza virus–specific IgG was measured by ELISA. n = 4 (−matAbs/C vaccine and −matAbs/S vaccine), n = 5 (naïve mice), n = 10 (+matAbs/PBS), n = 18 (+matAbs/C vaccine), or n = 19 (+matAbs/S vaccine) mice per group. (C) Mice (21 days old) were intramuscularly vaccinated with 1000 hemagglutination units of purified inactivated PR8 influenza virus (inact), 0.3 μg of PR8 influenza virus HA mRNA-LNP vaccine (mRNA), or PBS as control in the presence or absence of PR8 influenza virus–specific matAbs. Serum was collected 70 days post-vaccination, and influenza virus–specific serum IgG was measured by ELISA. Data are pooled from two independent experiments (n = 9 to 15 mice per group). Each point represents one mouse. Data are shown as means ± SD. Titers were compared by one-way ANOVA with Sidak’s (B) or Tukey’s (C) post hoc test. ****P < 0.0001. ns, not significant. Panel (B) shows results of two independent experiments. ns, not significant.
Fig. 5.. PR8 influenza virus HA mRNA-LNP vaccine elicits prolonged germinal center responses in the presence of matAbs.
(A) Flow cytometry gating strategy for hemagglutinin (HA)–positive germinal center (GC) B cells. (B and C) Mice were intramuscularly vaccinated with 1 μg of nucleoside-modified PR8 influenza virus HA mRNA-LNP vaccine, 1000 hemagglutination units of inactivated PR8 influenza virus, or 1 μg of poly(C) RNA-LNP at 21 days of age. Draining (popliteal) lymph nodes (B) and spleens (C) were collected, and HA-specific germinal center B cells (HA probe+ CD19+ B220+ CD138− PNA+ CD38−) were analyzed by flow cytometry. Data are pooled from three independent experiments. n = 4 (−matAbs) or n = 5 (+matAbs) mice per group per experiment at each time point. Each point represents one mouse. The line represents mean. Four- and 8-week post-vaccination time points within each condition were compared with the 2-week post-vaccination time point by one-way ANOVA with Dunnett’s post hoc test.
Fig. 6.. mRNA-LNP vaccine expressing Cal09 influenza virus strain hemagglutinin overcomes matAb inhibition.
(A) The experimental design is shown. (B) Serum was collected from mothers and pups on the day of weaning and A/California/07/2009 (Cal09) hemagglutinin (HA)–specific antibody titers were measured by hemagglutination inhibition. Each point represents one mouse. (C) Mice (21 days old) were vaccinated with human monovalent Cal09 influenza virus vaccine with MF59-like adjuvant (h + MF), 10 μg of mRNA-LNP vaccine expressing Cal09 HA (mRNA), PBS with MF59-like adjuvant (PBS + MF), or PBS alone (PBS) in the presence or absence of Cal09 HA-specific matAbs. Serum was collected 70 days post-vaccination, and Cal09 HA-specific serum IgG was measured by ELISA. Data are pooled from two independent experiments (n = 7 to 17 mice per group). Each point represents one mouse. Groups were compared by one-way ANOVA with Tukey’s post hoc test. ****P < 0.0001.
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