Impact of withholding breastfeeding around the time of Rotarix vaccination on the immunogenicity of Rotarix vaccine A Randomized Trial

Impact of withholding breastfeeding around the time of Rotarix vaccination on the immunogenicity of Rotarix vaccine A Randomized Trial S. Asad Ali, MD, MPH Department of Pediatrics and Child Health Aga Khan University Pakistan 1

Why is the immunogenicity of rotavirus vaccines lower in low income settings? 2

Why is the immunogenicity of rotavirus vaccines lower in low income settings? Environmental Enteropathy Higher anti-rv antibodies in mother s blood with transplacental transmission Higher anti-rv antibody levels in breast milk Concomitant OPV administration Coexistent intestinal infections 3

Why is the immunogenicity of rotavirus vaccines lower in low income settings? Environmental Enteropathy Higher anti-rv antibodies in mother s blood with transplacental transmission Higher anti-rv antibody levels in breast milk Concomitant OPV administration Coexistent intestinal infections 4

Moon et al. Page 8 FIGURE 1. Cumulative frequency profiles of rotavirus-specific antibodies in breast milk specimens from mothers in India, Vietnam, Korea and the United States. Milk specimens were tested for IgA (A) and neutralizing activity against vaccine strains Rotarix (B), RotaTeq G1 (C), Moon SS et al. Inhibitory effect of breast milk on infectivity of live oral rotavirus vaccines. Pediatr Infect Dis J. 2010 5

Why is the immunogenicity of rotavirus vaccines lower in low income settings? Environmental Enteropathy Higher anti-rv antibodies in mother s blood with transplacental transmission Higher anti-rv antibody levels in breast milk Concomitant OPV administration Coexistent intestinal infections 6

Primary Objective To compare the immunogenicity of Rotarix in infants whose breastfeeding was withheld one hour before through one hour after vaccination with each Rotarix dose to that in infants who were breastfed at the time of vaccination 7

Secondary Objective To determine the effect of rotavirus neutralizing activity in breast milk on the immunogenicity of Rotarix vaccine in infants 8

Trial profile Breas eeding immediately before RV administra on Randomiza on 1: 1 RV/B RV RV/B Breas eeding withheld for one hour before and a er RV administra on RV/B RV RV/B B B (n=200) (n=210) (n=200) (n=210) Birth 6 weeks 10 weeks 14 weeks 18 weeks RV = Rotarix Vaccine B = Blood Draw

Randomized (400) Breastfeed Withholding group(200) Immediate Breastfeeding group(200) IgA 20 U/mL (3) Non-compliance with blood draw (1) Lost to follow up/dropout Refusal (9) Illness (1) Moved from Study Area (5) IgA 20 U/mL (6) Non-compliance with blood draw (1) Non-compliance with breastfeeding schedule (5) Lost to follow up/dropout Refusal (9) Death (1) Moved from Study Area (6) Per Protocol (181) Per Protocol (172) 12

Baseline Characteristics and Age at visits Withholding Arm (N = 200) Immediate Breastfeed Arm (N = 200) Male 45 % 54 % Age in Weeks 6.3 (0.5) 6.3 (0.5) Weight (g) at 6 weeks 3939.1 (619.2) 4047.5 (594.7) Age of visit in weeks Visit 2 10.4 (0.5) 10.4 (0.6) Visit 3 14.6 (0.6) 14.6 (0.6) Visit 4 18.7 (0.7) 18.7 (0.7) per-protocol population 13

Anti-rotavirus IgA antibody seroconversion rates in the per protocol population Age at sample (weeks) 14 (after 2 doses) 18 (after 3 doses) Sero conversion rate Withholding Arm (N=181) 95 % CI Immediate Breastfeed Arm Sero conversion rate (N=172) 95 % CI P-Value 16.6% (11.8; 22.6) 29.1% (22.8; 36.2) 0.005 28.2% (22.1; 35.1) 37.8% (30.8; 45.2) 0.070

Anti-rotavirus IgA antibody seroconversion rates in the per protocol population Age at sample (weeks) 14 (after 2 doses) 18 (after 3 doses) Sero conversion rate Withholding Arm (N=181) 95 % CI Immediate Breastfeed Arm Sero conversion rate (N=172) 95 % CI P-Value 16.6% (11.8; 22.6) 29.1% (22.8; 36.2) 0.005 28.2% (22.1; 35.1) 37.8% (30.8; 45.2) 0.070

Anti-rotavirus IgA antibody seroconversion rates in the per protocol population Age at sample (weeks) 14 (after 2 doses) 18 (after 3 doses) Sero conversion rate Withholding Arm (N=181) 95 % CI Immediate Breastfeed Arm Sero conversion rate (N=172) 95 % CI P-Value 16.6% (11.8; 22.6) 29.1% (22.8; 36.2) 0.005 28.2% (22.1; 35.1) 37.8% (30.8; 45.2) 0.070

Anti-rotavirus IgA geometric mean titers (GMT) for seroconverted subjects after 2 or 3 doses Age at Sample (weeks) Withholding Arm Immediate Breastfeed Arm n GMT 95 % CI n GMT 95 % CI P value 14 (after 2 doses) a 30 194.4 (126.2;299.3) 50 104.4 (77.3; 140.9) 0.016 18 (after 3 doses) b 51 125.4 (91.0; 172.9) 65 91.8 (69.1; 121.8) 0.147 a Includes only anti-rotavirus IgA seropositive subjects at 14 weeks b Includes only anti-rotavirus IgA seropositive subjects at 18 weeks 17

Anti-rotavirus IgA geometric mean titers (GMT) for seroconverted subjects after 2 or 3 doses Age at Sample (weeks) Withholding Arm Immediate Breastfeed Arm n GMT 95 % CI n GMT 95 % CI P value 14 (after 2 doses) a 30 194.4 (126.2;299.3) 50 104.4 (77.3; 140.9) 0.016 18 (after 3 doses) b 51 125.4 (91.0; 172.9) 65 91.8 (69.1; 121.8) 0.147 a Includes only anti-rotavirus IgA seropositive subjects at 14 weeks b Includes only anti-rotavirus IgA seropositive subjects at 18 weeks 18

Anti-rotavirus IgA geometric mean titers (GMT) for seroconverted subjects after 2 or 3 doses Age at Sample (weeks) Withholding Arm Immediate Breastfeed Arm n GMT 95 % CI n GMT 95 % CI P value 14 (after 2 doses) a 30 194.4 (126.2;299.3) 50 104.4 (77.3; 140.9) 0.016 18 (after 3 doses) b 51 125.4 (91.0; 172.9) 65 91.8 (69.1; 121.8) 0.147 a Includes only anti-rotavirus IgA seropositive subjects at 14 weeks b Includes only anti-rotavirus IgA seropositive subjects at 18 weeks 19

Association of Maternally Derived Neutralizing Antibody (MDNA)* titers at 6 weeks with rotavirus seroconversion at 14 weeks (post 2 doses) MDNA at 6 weeks (Percentile) Withholding Arm Immediate Breastfeed Arm Combined (N= 93) (N= 85) (N= 169) Seroconversion rate Seroconversion rate Seroconversion rate < 25 22.5% 38.8% 31.4% > 75 5.7% 22.2% 12.4% P= 0.026 P= 0.157 P =0.003 *MDNA measured using 89:12 strain, which is precursor to Rotarix vaccine 20

Association of Maternally Derived Neutralizing Antibody (MDNA)* titers at 6 weeks with rotavirus seroconversion at 14 weeks (post 2 doses) MDNA at 6 weeks (Percentile) Withholding Arm Immediate Breastfeed Arm Combined (N= 93) (N= 85) (N= 169) Seroconversion rate Seroconversion rate Seroconversion rate < 25 22.5% 38.8% 31.4% > 75 5.7% 22.2% 12.4% P= 0.026 P= 0.157 P =0.003 *MDNA measured using 89:12 strain, which is precursor to Rotarix vaccine 21

Association of Maternally Derived Neutralizing Antibody (MDNA)* titers at 6 weeks with rotavirus seroconversion at 14 weeks (post 2 doses) MDNA at 6 weeks (Percentile) Withholding Arm Immediate Breastfeed Arm Combined (N= 93) (N= 85) (N= 169) Seroconversion rate Seroconversion rate Seroconversion rate < 25 22.5% 38.8% 31.4% > 75 5.7% 22.2% 12.4% P= 0.026 P= 0.157 P =0.003 *MDNA measured using 89:12 strain, which is precursor to Rotarix vaccine 22

Association of Maternally Derived Neutralizing Antibody (MDNA)* titers at 6 weeks with rotavirus seroconversion at 14 or 18 weeks (post 3 doses) MDNA at 6 weeks (Percentile) Withholding Arm Immediate Breastfeed Arm Combined (N= 93) (N= 85) (N= 169) Seroconversion rate Seroconversion rate Seroconversion rate < 25 37.5% 46.9% 42.7% > 75 11.3% 19.5% 14.6% P= 0.005 P= 0.012 P <0.0001 *MDNA measured using 89:12 strain, which is precursor to Rotarix vaccine 23

Association of Maternally Derived Neutralizing Antibody (MDNA)* titers at 6 weeks with rotavirus seroconversion at 14 or 18 weeks (post 3 doses) MDNA at 6 weeks (Percentile) Withholding Arm Immediate Breastfeed Arm Combined (N= 93) (N= 85) (N= 169) Seroconversion rate Seroconversion rate Seroconversion rate < 25 37.5% 46.9% 42.7% > 75 11.3% 19.5% 14.6% P= 0.005 P= 0.012 P <0.0001 *MDNA measured using 89:12 strain, which is precursor to Rotarix vaccine 24

Association of Maternally Derived Neutralizing Antibody (MDNA)* titers at 6 weeks with rotavirus seroconversion at 14 or 18 weeks (post 3 doses) MDNA at 6 weeks (Percentile) Withholding Arm Immediate Breastfeed Arm Combined (N= 93) (N= 85) (N= 169) Seroconversion rate Seroconversion rate Seroconversion rate < 25 37.5% 46.9% 42.7% > 75 11.3% 19.5% 14.6% P= 0.005 P= 0.012 P <0.0001 *MDNA measured using 89:12 strain, which is precursor to Rotarix vaccine 25

Serum Anti-Rotavirus IgA Seroconversion in Immediate Breastfeeding Group, by MDNA and 10-Week BMNA Level Time point Low BMNA in Breast Milk at 10 weeks High BMNA in Breast Milk at 10 weeks (<50 %ile) (>50 %ile) MDNA at 6 weeks Seroconversion rate Seroconversion rate Difference in seroconversion P-value 14 weeks (post 2 doses) <50 %ile 44.4% 20% 24.4% 0.019 >50 %ile 29% 27.3% 1.8% 1 18 weeks (post 3 doses) <50 %ile 38.9% 38% 0.9% 1 >50 %ile 45.2% 32.7% 12.4% 0.353 MDNA Maternally derived neutralizing antibodies; BMNA Breast milk neutralizing activity 26

Serum Anti-Rotavirus IgA Seroconversion in Immediate Breastfeeding Group, by MDNA and 10-Week BMNA Level Time point Low BMNA in Breast Milk at 10 weeks High BMNA in Breast Milk at 10 weeks (<50 %ile) (>50 %ile) MDNA at 6 weeks Seroconversion rate Seroconversion rate Difference in seroconversion P-value 14 weeks (post 2 doses) <50 %ile 44.4% 20% 24.4% 0.019 >50 %ile 29% 27.3% 1.8% 1 18 weeks (post 3 doses) <50 %ile 38.9% 38% 0.9% 1 >50 %ile 45.2% 32.7% 12.4% 0.353 MDNA Maternally derived neutralizing antibodies; BMNA Breast milk neutralizing activity 27

Serum Anti-Rotavirus IgA Seroconversion in Immediate Breastfeeding Group, by MDNA and 10-Week BMNA Level Time point Low BMNA in Breast Milk at 10 weeks High BMNA in Breast Milk at 10 weeks (<50 %ile) (>50 %ile) MDNA at 6 weeks Seroconversion rate Seroconversion rate Difference in seroconversion P-value 14 weeks (post 2 doses) <50 %ile 44.4% 20% 24.4% 0.019 >50 %ile 29% 27.3% 1.8% 1 18 weeks (post 3 doses) <50 %ile 38.9% 38% 0.9% 1 >50 %ile 45.2% 32.7% 12.4% 0.353 MDNA Maternally derived neutralizing antibodies; BMNA Breast milk neutralizing activity 28

Summary of Findings Withholding breast feeding around the time of RV1 vaccine administration did not lead to increased anti-rotavirus IgA seroconversion. To our surprise, IgA seroconversion after two RV1 doses at 6 and 10 weeks was significantly greater in immediately breastfed infants compared with those in whom breast milk was withheld! 29

Some nuances! GMT in seropositive infants at 14 weeks was higher in the withholding arm compared to the immediate arm Lower seroconversion in infants in the immediate feeding arm with low MDNA/higher BMNA after two doses suggests that ingesting breast milk with higher BMNA at the same time as vaccine may reduce the immune response in some infants 30

Summary Although serum rotavirus IgA is not a perfect correlate of protection, our findings suggest that substantially improved clinical protection is not likely with the strategy of withholding breastfeed around the time of vaccination. We suggest breastfeeding can continue ad lib around the time of RV1 administration. 31

But why was serum rotavirus IgA seroconversion higher in the immediate breastfeed group? Chance finding? Additional buffering of gastric acid at the time of vaccine administration? Something immunogenic in breast milk?? 32

Aga Khan University Study Team: PATH CDC Asad Ali (PI) Momin Kazi Anita Zaidi Rota study team Jessica Fleming Chris Victor Kathy Neuzil Duncan Steele Margaret Cortese Umesh Parashar Baoming Jia SungSil Moon Patients and their families University of Cincinnati Monica McNeal