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A polyvalent inactivated rhinovirus vaccine is broadly immunogenic in rhesus macaques

Immunogenicity of 10-valent HRV vaccine in mice

We first used BALB/c mice to test immunogenicity. We propagated HRVs in H1-HeLa cells and inactivated infectivity using formalin. Sera from naive mice had no detectable nAb against HRV-16 (Fig. 1). Alum adjuvant enhanced the nAb response induced by i.m. inactivated HRV-16 (Fig. 1). There was no effect of valency (comparing 1-, 3-, 5-, 7- and 10-valent) on the nAb response induced by inactivated HRV-16 or to the 3 types in the 3-valent vaccine (HRV-16, HRV-36 and HRV-78; Fig. 1). The 50% tissue culture infectious dose (TCID50) titres of the input viruses before inactivation (inactivated-TCID50) are provided in Supplementary Table 1. Original antigenic sin can occur when sequential exposure to related virus variants results in biased immunity to the type encountered first32. In bivalent HRV-immune mice, we observed modest original antigenic sin following prime vaccination with 10-valent inactivated HRV, and boost vaccination partially alleviated the effect (Supplementary Fig. 1), similar to influenza virus32. Collectively, these results prompted us to explore more fully the nAb response to polyvalent HRV vaccine.

Sufficient input HRV titre determines immunogenicity

In 1975, it was reported that two different 10-valent inactivated HRV preparations induced nAb titres to only 30–40% of the input virus types in recipient subjects33. However, the input titres of viruses before inactivation ranged from 101.5 to 105.5 TCID50 per ml, and these were then diluted 10-fold to generate 10-valent 1.0 ml doses given i.m. as prime and boost with no adjuvant33. We hypothesized that low input antigen doses are responsible for poor nAb responses to 10-valent inactivated HRV. We reconstituted the 1975 10-valent vaccine, as closely as possible with available HRV types, over a 101 to 105 inactivated-TCID50 per vaccine dose, and we compared it with a 10-valent vaccine of the same types with input titres ranging from>105 to>107 inactivated-TCID50 per dose. The reconstituted 1975 vaccine resulted in no detectable nAb after prime vaccination and, following boost vaccination, nAb to the five types that had the highest input titres (Fig. 2). The high titre vaccine resulted in nAb to 5 of 10 types after prime vaccination and all 10 types after the boost (Fig. 2). Following the boost vaccinations, there appeared to be a 104 inactivated-TCID50 per vaccine dose threshold for the induction of nAb in this model (Fig. 2b). Above this titre, there was no correlation between input load and nAb induction.

Immunogenicity of 25-valent HRV vaccine in mice

Injectable vaccines used in people are commonly given in a 0.5 ml dose. In our facility, the highest allowable i.m. vaccine volume in mice was 0.1 ml. We tested a 25-valent per 0.1 ml HRV vaccine in mice as a scalable prototype. The 25-valent inactivated HRV vaccine had a 7.4-fold lower average inactivated-TCID50 per type per dose than the 10-valent composition (Supplementary Table 2) to accommodate the volume adjustment. The 10-valent inactivated HRV vaccine induced nAb to 100% of input types following the prime and the boost (Fig. 3a). The nAb induced by 10-valent inactivated HRV were persisting at 203 days post-boost (Supplementary Fig. 2). We used two different sets of virus stocks for the prime and boost 25-valent vaccinations in these experiments (Supplementary Table 2). The reason is that in the interim between the prime and boost vaccinations, we obtained higher titre virus stocks of eleven of the input types (HRV-32, -49, -58, -55, -41, -33, -39, -50, -1B, -21 and -60). The higher titre stocks of these eleven were used in the boost vaccination. The 25-valent inactivated HRV prime vaccination induced nAb to 18 of 25 (72%) virus types, and the 25-valent boost resulted in nAb against 24 of the 25 types (96%; Fig. 3b). The average nAb titre resulting from prime and boost was 27 for 10-valent and 26.8 for 25-valent. These data demonstrate broad neutralization of diverse HRV types with a straightforward vaccine approach.

Immunogenicity of 25- and 50-valent HRV in RMs

To increase vaccine valency, we chose RMs and a 1.0 ml i.m. vaccine volume. Two RMs were vaccinated with 25-valent inactivated HRV, and two RMs were vaccinated with 50-valent inactivated HRV. Pre-immune sera in RM A and RM B had no detectable nAb against the 25 HRV types included in the 25-valent vaccine. The inactivated-TCID50 titres per dose were higher in RMs than in mice (Supplementary Table 3). The 25-valent vaccine induced nAb to 96% (RM A) and 100% (RM B) of input viruses following the prime vaccination (Fig. 4a). The 50-valent vaccine induced nAb to 90% (RM C) and 82% (RM D) of input viruses following the prime vaccination (Fig. 4c). The breadth of nAb following prime vaccination in RM was superior to what we observed in mice, which may have been due to animal species differences and/or higher inactivated-TCID50 input titres in the RM vaccines. Following boost vaccination, there were serum nAb titres against 100% of the types in 25-valent HRV-vaccinated RMs (Fig. 4b) and 98% (49 out of 50) of the virus types in 50-valent HRV-vaccinated RMs (Fig. 4d). The average nAb titre resulting from prime and boost in RMs was 29.3 for 25-valent and 28.6 for 50-valent. The nAb responses were type-specific, not cross-neutralizing, because there were minimal nAbs induced by the 25-valent vaccine against 10 non-vaccine types (Supplementary Fig. 3). The nAb response to 50-valent inactivated HRV vaccine was broad and potent in RMs.

HRV yield in WI-38 cells and semi-purification by HPLC

The HRV stocks used in our vaccinations were produced in H1-HeLa cells, a good substrate for HRV replication but not suitable for vaccine manufacturing. We compared the infectious yield of 10 HRV types in H1-HeLa and WI-38, which can be qualified for vaccine production. Adequate yields were obtained from WI-38 cells (Supplementary Fig. 4). Injectable vaccines require defined purity. As proof of principle, we semi-purified three HRV types by high-performance liquid chromatography and found uncompromised immunogenicity of trivalent inactivated semi-purified HRV in mice (Supplementary Fig. 5).


In this study, we demonstrate that polyvalent inactivated HRV vaccine expanded to a 50-valent composition, with alum adjuvant, is immunogenic against approximately one-third of circulating HRV types. Although fifty HRV types is a minority, these data provide proof of principle for a highly polyvalent vaccine approach. Forty years ago, the prospects for a polyvalent HRV vaccine were dour for good reasons17. However, progress in technology34 and advancement of more complex vaccines renders impediments to a polyvalent HRV vaccine manageable. Scale-up of HRV vaccines may be facilitated by related vaccine production processes and new cost-saving manufacturing technologies353637. Inactivated HRV has a positive history of clinical efficacy689.

On the basis of our results and doses of early immunogenic HRV vaccines68, we estimate 104–5 inactivated-TCID50 per type per dose will be useful. Therefore, HRV stock titres≥107 TCID50 per ml are required for a potential 83-valent HRV A formulation in a 0.5 ml dose containing alum adjuvant. In future studies, we hope to produce a comprehensive 83-valent HRV A vaccine. A limitation of our study is that we have not included HRV C antigens, which will be important for HRV vaccine development, especially for paediatric populations. Ongoing efforts are aimed at propagation of HRV C strains to high titre in cell lines and development of serology assays. Ultimately, our approach may lead to vaccines for rhinovirus-mediated diseases including asthma and COPD exacerbations and the common cold. Advancing valency may be applicable to vaccines for other antigenically variable pathogens.

Cell lines and viruses

H1-HeLa (CRL-1958) and WI-38 (CCL-75) cells were obtained from the American Type Culture Collection (ATCC) and cultured in minimal essential media with Richter's modification and no phenol red (MEM) (Thermo Fisher) supplemented with 10% fetal bovine serum. The cell lines were not authenticated but are not commonly misidentified (International Cell Line Authentication Committee). We tested HeLa-H1 cells using the LookOut Mycoplasma detection kit (Sigma), and these were mycoplasma negative. HRV-7 (VR-1601), HRV-9 (VR-1745), HRV-11 (VR-1567), HRV-13 (VR-286), HRV-14 (VR-284), HRV-16 (VR-283), HRV-19 (V4-1129), HRV-24 (VR-1134), HRV-29 (VR-1809), HRV-30 (VR-1140), HRV-31 (VR-1795), HRV-32 (VR-329), HRV-36 (VR-509), HRV-38 (VR-511), HRV-40 (VR-341), HRV-41 (VR-1151), HRV-49 (VR-1644), HRV-53 (VR-1163), HRV-56 (VR-1166), HRV-58 (VR-1168), HRV-59 (VR-1169), HRV-60 (VR-1473), HRV-64 (VR-1174), HRV-66 (VR-1176), HRV-68 (VR-1178), HRV-75 (VR-1185), HRV-76 (VR-1186), HRV-77 (VR-1187), HRV-78 (VR-1188), HRV-80 (VR-1190), HRV-81 (VR-1191), HRV-85 (VR-1195), HRV-88 (VR-1198), HRV-89 (VR-1199), HRV-96 (VR-1296), and HRV-100 (VR-1300) prototype strains were purchased from ATCC. HRV-1B, HRV-10, HRV-21, HRV-28, HRV-33, HRV-34, HRV-39, HRV-45, HRV-50, HRV-51, HRV-54, HRV-55 and HRV-94 strains were obtained from the Centers for Disease Control and Prevention. The HRVs in the study are species A, with the exception of HRV-14 (B), and represent A species broadly2021.

HRV propagation and titration

HRV stocks were generated in H1-HeLa cells. Approximately 0.5 ml of HRV was inoculated onto subconfluent H1-HeLa monolayer cells in a T-182 flask. After adsorption for 1 h at room temperature with rocking, 50 ml of HRV infection medium (MEM supplemented with 2% FBS, 20 mM HEPES, 10 mM MgCl2, 1 × non-essential amino acids (Gibco catalog 11140-050)) was added and the infection was allowed to proceed at 32 °C in a 5% CO2 humidified incubator until the monolayer appeared to be completely involved with cytopathic effect (CPE), 1 to 3 days post-infection. The cells were scraped, and the cells and medium (∼50 ml) were transferred to two pre-chilled 50 ml conical polypropylene tubes and kept on ice while each suspension was sonicated using a Sonic Dismembrator Model 500 (Fisher Scientific) equipped with a ½-inch diameter horn disrupter and 1/4-inch diameter tapered microtip secured on a ring stand. Sonication was performed by an operator in a closed room with ear protection, at 10% amplitude, 1 s on/1 s off intervals and 1 pulse per 1 ml of material. Sonication yielded higher titres than freeze–thaw. The suspension was clarified by centrifugation at 931g for 10 min. The supernatant was transferred to cryovials, snap-frozen in liquid nitrogen and stored at −80 °C. For comparing HRV yield in H1-HeLa and WI-38 cells, T-75 flasks of subconfluent cells were infected at a multiplicity of infection of 0.1 TCID50 per cell, and 20 ml of culture medium were discarded before scraping the cells in the remaining 5 ml followed by sonication. For all stocks, TCID50 ml−1 titres were determined by infecting subconfluent H1-HeLa cells in 96-well plates with serially diluted samples, staining the cells 6 days post-infection with 0.1% crystal violet/20% methanol, scoring wells for CPE and calculating the end point titre using the Reed and Muench method38.

HRV purification

HRV stock was collected from H1-HeLa cell monolayers as described above and clarified by brief centrifugation at low speed to remove large cellular debris (931g, 10 min, 4 °C). To remove excess albumin from the crude virus stock by affinity chromatography, the supernatant was loaded onto a HiTrap Blue HP column (GE Healthcare) using an ÄKTA Purifier system (GE Healthcare) according to the manufacturer specifications. Flowthrough was subsequently loaded through a HiTrap Capto Core 700 column (GE Healthcare) to refine the virus prep by size exclusion chromatography. The flowthrough from the HiTrap Blue HP and the HiTrap Capto Core 700 was captured using the ÄKTA Purifier system (GE Healthcare) with a 20 mM sodium phosphate buffer (pH 7.0). Flowthrough from size exclusion chromatography was dialyzed overnight with 0.1 M Tris-HCl buffer (pH 8.0), then loaded onto a HiTrap Q XL column (GE Healthcare) and separated into fractions by ion exchange chromatography. Virus-containing fractions were eluted using the ÄKTA Purifier system (GE Healthcare) with a 0.1 M Tris-HCl buffer (pH 8.0) and a sodium chloride gradient. Fractions showing high absorption peaks at 280 nM were collected and analysed for viral titre by TCID50 end point dilution assay, and fraction purity visualized on a 10% SDS-PAGE gel by silver stain (Thermo Fisher Scientific; Supplementary Fig. 6 and Supplementary Table 4). Fractions of HRV-16, HRV-36 and HRV-78 of high virus titre and purity were combined for formalin inactivation as described below.

Mice and RMs

All experiments involving animals were performed at Emory University and the Yerkes National Primate Research Center in accordance with guidelines established by the Animal Welfare Act and the NIH Guide for the Care and Use of Laboratory Animals. Animal facilities at Emory University and the Yerkes Center are fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). The Institutional Animal Care and Use Committee (IACUC) of Emory University approved these studies. Pathogen-free, 6–7-week female BALB/c mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Mice were randomly assigned to groups based on the sequential selection from an inventory, and investigators were not blinded to outcome assessment. No statistical methods were used in predetermining sample sizes.

Young adult (3–5 kg, 2–4 years of age, 2 females and 2 males) Indian RMs (Macaca mulatta; RM) were maintained according to NIH guidelines at the Yerkes National Primate Research Center. Handling and movement of RMs was performed by qualified personnel who have received specific training in safe and humane methods of animal handling at the Yerkes National Primate Research Center. The initial exclusion criterion was pre-existing nAb against HRV. The studies were performed in strict accordance with US Department of Agriculture regulations and the recommendations in the Guide for Care and Use of Laboratory Animals of the NIH. The RMs were allocated in an un-blinded manner to two vaccine groups (25-valent and 50-valent), one male and one female per group. No statistical methods were used in predetermining sample sizes.


Before immunization, all HRV types were inactivated by addition of 0.025% formalin followed by incubation with stirring for 72 h at 37 °C, as previously described for HRV vaccine33. Complete inactivation of infectivity was confirmed by end point TCID50 titration in H1-HeLa cells. Formalin inactivation by this method resulted in greater immunogenicity in mice than alternative inactivation by β-propiolactone, suggesting formalin inactivation preserved antigenic determinants. Mice were vaccinated i.m. with inactivated HRV strains mixed with 100 μg of Alhydrogel adjuvant 2% (aluminium hydroxide wet gel suspension, alum; Sigma catalog A8222 or Invivogen catalog vac-alu) according to instructions of the manufacturers. The total volume per mouse was 100 μl, administered in 50 μl per thigh. Mice were given a second identical vaccination (boost) at the time indicated in figure legends. RMs were vaccinated i.m. with inactivated HRV strains mixed with 500 μg of Alhydrogel adjuvant 2%. The total volume per RM was 1 ml, administered in one leg. RMs were boosted with an identical vaccination at four weeks.

Serum collection

In mice, peripheral blood was collected into microcentrifuge tubes from the submandibular vein. Samples were incubated at room temperature for 20 min to clot. The tubes were centrifuged 7,500g for 10 min to separate serum. The serum samples were pooled from mice of each group and stored at −80 °C until used. Phlebotomy involving RMs was performed under either ketamine (10 mg kg−1) or Telazol (4 mg kg−1) anaesthesia on fasting animals. Following anaesthesia with ketamine or Telazol, the animals were bled from the femoral vein. Yerkes blood collection guidelines were followed and no more than 10 ml kg−1 per 28 days of blood was collected. After collecting blood in serum separating tube, samples were incubated at room temperature for 30 min. The tubes were centrifuged at 2,500g for 15 min to separate serum. The serum samples from individual RM were stored at −80 °C until used.

Serum neutralization assay

H1-HeLa cells were seeded in 96-well plates to attain 80–90% confluence in 24 h. Due to the limited volume of mouse serum per bleed and the number of nAb assays required, we pooled mouse sera. After separating serum from whole blood, equal volumes of sera per mouse were pooled from each group, before heat inactivation. Levels of nAb against the indicated HRV types in the vaccines used in mice were measured in pooled sera. Heat-inactivated (56 °C, 30 min) serum samples were twofold serially diluted in MEM and added to 500 TCID50 ml−1 HRV of each type to be tested, in an equal volume. The virus and serum mixtures were incubated at 37 °C for 1 h. Then, 50 μl of the serum–virus mixture was transferred onto H1-HeLa cell monolayers in 96-well plates in triplicate, and plates were spinoculated at 2,095g for 30 min at 4 °C. For each type, a no-serum control was added to test the input 500 TCID50. We tested pooled HRV-16 anti-sera against HRV-16 in each assay as a standard. After spinoculation, 150 μl of HRV infection medium were added to each well. The 96-well plates were incubated for 6 days at 32 °C and 5% CO2 and then stained with crystal violet as described above. Wells were scored for the presence or absence of CPE. Neutralizing antibody end point titres and 95% confidence intervals were determined by the method of Reed and Muench38, as previously described for HRV1438. The 95% confidence interval indicates variability of three technical replicates within a single nAb experiment. An example of the nAb assay and titre calculation is provided in Supplementary Fig. 7.

Data availability

Primary data that support the findings of this study are available from the corresponding author on request.

Additional information

How to cite this article: Lee, S. et al. A polyvalent inactivated rhinovirus vaccine is broadly immunogenic in rhesus macaques. Nat. Commun. 7:12838 doi: 10.1038/ncomms12838 (2016).